Cellulase variants with improved expression, activity and stability, and use thereof

ABSTRACT

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having improved expression, activity and/or stability. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, and methods of use thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/183,959, filed on Jun. 3, 2009, which is hereby incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under conditional award no: DE-FC36-08GO18078 awarded by the Department of Energy. The Government has certain rights in this invention.

I. FIELD

The present disclosure relates to enzymes and in particular cellulase variants. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, methods of identifying additional useful cellulase variants and methods of use thereof.

II. BACKGROUND

Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded and used as an energy source by numerous microorganisms (e.g., bacteria, yeast and fungi) that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., J Biol Chem, 276: 24309-24314, 2001). As the limits of non-renewable resources approach, the potential of cellulose to become a major renewable energy resource is enormous (Krishna et al., Bioresource Tech, 77: 193-196, 2001). The effective utilization of cellulose through biological processes is one approach to overcoming the shortage of foods, feeds, and fuels (Ohmiya et al., Biotechnol Gen Engineer Rev, 14: 365-414, 1997).

Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91) (“CBH”) and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC 3.2.1.21) (“BG”) (Knowles et al., TIBTECH 5: 255-261, 1987; and Schulein, Methods Enzymol, 160: 234-243, 1988). Endoglucanases act mainly on the amorphous parts of the cellulose fibre, whereas cellobiohydrolases are also able to degrade crystalline cellulose (Nevalainen and Penttila, Mycota, 303-319, 1995). Thus, the presence of a cellobiohydrolase in a cellulase system is required for efficient solubilization of crystalline cellulose (Suurnakki et al., Cellulose 7: 189-209, 2000). Beta-glucosidase acts to liberate D-glucose units from cellobiose, cello-oligosaccharides, and other glucosides (Freer, J Biol Chem, 268: 9337-9342, 1993).

Cellulases are known to be produced by a large number of bacteria, yeast and fungi. Certain fungi produce a complete cellulase system capable of degrading crystalline forms of cellulose, such that the cellulases are readily produced in large quantities via fermentation. Filamentous fungi play a special role since many yeast, such as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose (See, e.g., Wood et al., Methods in Enzymology, 160: 87-116, 1988).

The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. For example, multiple CBHs, EGs and BGs have been isolated from a variety of fungal sources including Trichoderma reesei (also referred to as Hypocrea jecorina), which contains known genes for two CBHs, i.e., CBH I (“CBH1”) and CBH II (“CBH2”), at least 8 EGs, i.e., EG I, EG II, EG III, EGIV, EGV, EGVI, EGVII and EGVIII, and at least 5 BGs, i.e., BG1, BG2, BG3, BG4 and BG5. EGIV, EGVI and EGVIII also have xyloglucanase activity.

In order to efficiently convert crystalline cellulose to glucose the complete cellulase system comprising components from each of the CBH, EG and BG classifications is required, with isolated components less effective in hydrolyzing crystalline cellulose (Filho et al., Can J Microbiol, 42:1-5, 1996). A synergistic relationship has been observed between cellulase components from different classifications. In particular, the EG-type cellulases and CBH-type cellulases synergistically interact to more efficiently degrade cellulose.

Cellulases are known in the art to be useful in the treatment of textiles for the purposes of enhancing the cleaning ability of detergent compositions, for use as a softening agent, for improving the feel and appearance of cotton fabrics, and the like (Kumar et al., Textile Chemist and Colorist, 29:37-42, 1997). Cellulase-containing detergent compositions with improved cleaning performance (U.S. Pat. No. 4,435,307; GB App. Nos. 2,095,275 and 2,094,826) and for use in the treatment of fabric to improve the feel and appearance of the textile (U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757; and GB App. No. 1,358,599), have been described. Hence, cellulases produced in fungi and bacteria have received significant attention. In particular, fermentation of Trichoderma spp. (e.g., Trichoderma longibrachiatum or Trichoderma reesei) has been shown to produce a complete cellulase system capable of degrading crystalline forms of cellulose.

Although cellulase compositions have been previously described, there remains a need for new and improved cellulase compositions. Improved cellulose compositions find used in household detergents, textile treatments, biomass conversion and paper manufacturing. Cellulases that exhibit improved expression, activity and stability are of particular interest.

III. SUMMARY

The present disclosure relates to enzymes and in particular cellulase variants. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, methods of identifying additional useful cellulase variants and methods of use thereof.

The present disclosure provides cellulase variants, wherein the variants are mature forms having cellulase activity and comprising a substitution at one or more positions selected from the group consisting of: 5, 18, 19, 28, 30, 32, 35, 38, 79, 80, 89, 100, 102, 103, 104, 105, 111, 117, 119, 121, 125, 126, 133, 137, 138, 139, 140, 141, 143, 150, 158, 162, 177, 180, 181, 182, 185, 186, 188, 190, 191, 192, 193, 196, 201, 207, 225, 226, 228, 229, 230, 233, 234, 236, 240, 243, 245, 251, 252, 258, 267, 268, 274, 292, 293, 303, 304, 306, 307, 313, 319, 322, 328, 331, 338, 340, 346, 361, 362, 363, 364, 365, 371, 384, 394, 396, 400, 406, 407, 414, 417, 422, 427, 431, 433, 436, 440, 441, 443, 444, 445 and 447, wherein the positions are numbered by correspondence with the amino acid sequence of a reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3. In some embodiments, the substitution at one or more positions results in a cellulase variant with improved expression, activity and/or stability in comparison to the reference CBH2. In some embodiments, the variant comprises a further substitution at one or more further positions selected from: (i) a first group consisting of 63, 77, 129, 146, 147, 151, 153, 157, 161, 189, 194, 197, 203, 204, 208, 211, 237, 239, 244, 247, 254, 277, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, 382 and 405; or (ii) a second group consisting of 94, 98, 107, 120, 134, 144, 147, 154, 178, 179, 206, 210, 214, 231, 232, 266, 272, 275, 316, 323, 343, 360, 380, 381, 386, 399, 410, 413, 416, 426, and 429, wherein the further positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3. In some embodiments, the substitution at one or more positions is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 positions.

In another aspect the disclosure provides cellulase variants, wherein the variants are mature forms having cellulase activity and comprising a substitution at one or more positions selected from the group consisting of: 63, 77, 129, 146, 147, 151, 153, 157, 161, 189, 194, 197, 203, 204, 208, 211, 237, 239, 244, 247, 254, 277, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, 382 and 405, wherein the positions are numbered by correspondence with the amino acid sequence of a reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3 and wherein when the cellulase variant consists of a single substitution, the single substitution is not a K/E, R/Q, N/D, Q/E, D/N or E/Q substitution. In some embodiments, the substitution at one or more positions results in a cellulase variant with improved expression, activity and/or stability in comparison to the reference CBH2. In some embodiments, the isolated cellulase variant comprises a further substitution at one or more further positions selected from: (i) a first group consisting of 5, 18, 19, 28, 30, 32, 35, 38, 79, 80, 89, 100, 102, 103, 104, 105, 111, 117, 119, 121, 125, 126, 133, 137, 138, 139, 140, 141, 143, 150, 158, 162, 177, 180, 181, 182, 185, 186, 188, 190, 191, 192, 193, 196, 201, 207, 225, 226, 228, 229, 230, 233, 234, 236, 240, 243, 245, 251, 252, 258, 267, 268, 274, 292, 293, 303, 304, 306, 307, 313, 319, 322, 328, 331, 338, 340, 346, 361, 362, 363, 364, 365, 371, 384, 394, 396, 400, 406, 407, 414, 417, 422, 427, 431, 433, 436, 440, 441, 443, 444, 445 and 447; or (ii) a second group consisting of 94, 98, 107, 120, 134, 144, 147, 154, 178, 179, 206, 210, 214, 231, 232, 266, 272, 275, 316, 323, 343, 360, 380, 381, 386, 399, 410, 413, 416, 426, and 429, wherein the further positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3. In some embodiments, the substitution at one or more positions is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 positions.

In another aspect the disclosure provides cellulase variants, wherein the variants are mature forms having cellulase activity and comprising a substitution at one or more positions selected from the group consisting of: 94, 98, 107, 120, 134, 144, 147, 154, 178, 179, 206, 210, 214, 231, 232, 266, 272, 275, 316, 323, 343, 360, 380, 381, 386, 399, 410, 413, 416, 426, and 429, wherein the positions are numbered by correspondence with the amino acid sequence of a reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not one of the group consisting of V94E, P98L, E107Q, M120L, M134G, M134L, M134V, L144G, L144R, L144S, T154A, A178V, L179C, V206L, S210L, S210R, T214M, T214Y, G231P, G231I, G231A, G231N, G231S, G231T, T232V, H266S, H266A, W272A, W272D, W272Y, N275L, S316P, V323L, V323N, V323Y, G360R, S380T, A381T, E399N, E399D, R413Y, R413P, and A416G. In some embodiments, the substitution at one or more positions results in a cellulase variant with improved expression, activity and/or stability in comparison to the reference CBH2. In some embodiments, the variant comprises a further substitution at one or more further positions selected from: (i) a first group consisting of 5, 18, 19, 28, 30, 32, 35, 38, 79, 80, 89, 100, 102, 103, 104, 105, 111, 117, 119, 121, 125, 126, 133, 137, 138, 139, 140, 141, 143, 150, 158, 162, 177, 180, 181, 182, 185, 186, 188, 190, 191, 192, 193, 196, 201, 207, 225, 226, 228, 229, 230, 233, 234, 236, 240, 243, 245, 251, 252, 258, 267, 268, 274, 292, 293, 303, 304, 306, 307, 313, 319, 322, 328, 331, 338, 340, 346, 361, 362, 363, 364, 365, 371, 384, 394, 396, 400, 406, 407, 414, 417, 422, 427, 431, 433, 436, 440, 441, 443, 444, 445 and 447; or (ii) a second group consisting of 63, 77, 129, 146, 147, 151, 153, 157, 161, 189, 194, 197, 203, 204, 208, 211, 237, 239, 244, 247, 254, 277, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, 382 and 405, wherein the further positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3. In some embodiments, the substitution at one or more positions is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 positions.

The present disclosure further provides cellulase variants, wherein the variants are mature forms having cellulase activity and comprising a substitution at from one to six positions selected from the group consisting of 111, 144, 154, 162, 410 and 413, wherein the cellulase variant comprises: a leucine or a serine at position 111; a leucine, a glutamine or a tryptophan at position 144; a threonine, a cysteine or a valine at position 154; a tyrosine or an asparagine at position 162; an arginine or a serine at position 410; and a serine, a tryptophan or a tyrosine at position 413; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not a S413Y substitution.

Also provided by the present disclosure are cellulase variants, wherein the variants are mature forms having cellulase activity and comprising a substitution at from one to six positions selected from the group consisting of 98, 194, 313, 316, 384 and 443, wherein the cellulase variant comprises: a proline or a leucine at position 98; a lysine, a cysteine or a glutamic acid at position 194; a serine or a threonine at position 313; a serine or a proline at position 316; a glycine, a cysteine or a glutamine at position 384; and an asparagine or an isoleucine at position 443; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not one of the group selected from a P98L substitution, a K194E substitution, and a S316P substitution.

In addition the present disclosure provides cellulase variants, wherein the variants are mature forms having cellulase activity and comprising a substitution at from one to six positions selected from the group consisting of 153, 161, 203, 233, 422 and 444, wherein the cellulase variant comprises: an arginine or a glutamine at position 153; an asparagine, an alanine or a tryptophan at position 161; an arginine or a histidine at position 203; a proline or an aspartic acid at position 233; a glutamine or a valine at position 422; and a proline or a glutamine at position 443; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not a R153Q substitution.

Moreover the present disclosure provides cellulase variants, wherein the variants are mature forms having cellulase activity and comprising a substitution at from one to seven positions selected from the group consisting of 98, 111, 144, 313, 316, 413 and 422, wherein the cellulase variant comprises: a proline or a leucine at position 98; a leucine or a serine at position 111; a leucine or a tryptophan at position 144; a serine or a threonine at position 313; a serine or a proline at position 316; a serine or a tryptophan at position 413; and a glutamine or a valine at position 422; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not one of the group selected from a P98L substitution, and a S316P substitution.

In further aspects of the disclosure cellulase variants are provided, wherein the variants comprise a glutamine at position 98, and a substitution selected from the group consisting of a T138C, a S316P, a S343Q, a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.

Additionally, cellulase variants are provided which comprise a cysteine at position 138, and a substitution selected from the group consisting of a S316P, a S343Q, a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.

In another aspect, cellulase variants are provided which comprise a proline at position 316, and a substitution selected from the group consisting of a S343Q, a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.

Moreover, the present disclosure provides cellulase variants comprising a glutamine at position 343, and a substitution selected from the group consisting of a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.

The present disclosure also provides cellulase variants comprising an isoleucine at position 362, and a substitution selected from the group consisting of a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.

In one aspect, cellulase variants are provided comprising a cysteine at position 386, and a substitution selected from the group consisting of a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3. In some embodiments, the variants comprise a serine at position 400 and a proline at position 406.

In addition, the present disclosure provides cellulase variants, wherein the variant is a mature form having cellulase activity and comprising a substitution at from one to seven positions selected from the group consisting of 98, 111, 182, 291, 316, 362 and 400, wherein the cellulase variant comprises: proline, leucine or glutamine at position 98; leucine or serine at position 111; aspargine or tryptophan at position 182; serine or a glutamic acid at position 291; serine or proline at position 316; glutamine, isoleucine or leucine at position 362; and cysteine or serine at position 400; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not a P98L substitution or a S316P substitution. In some embodiments, the variant further comprises a L439P substitution or a T74S substitution.

In some preferred embodiments of the disclosure, the cellulase variants of any of the preceding paragraphs of the summary are derived from a parent cellulase selected from the group consisting of Hypocrea jecorina CBH2, Hypocrea koningii CBH2, Humicola insolens CBH2, Acremonium cellulolyticus CBH2, Agaricus bisporus CBH2, Fusarium osysporum EG, Phanerochaete chrysosporium CBH2, Talaromyces emersonii CBH2, Thermobifida. fusca 6B/E3 CBH2, Thermobifida fusca 6A/E2 EG, and Cellulomonas fimi CenA EG. In some embodiments, the cellulase variant is derived from a parent cellulase whose amino acid sequence is at least 75% 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a member of the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In some embodiments, the variants are isolated. Also provided are compositions comprising a cellulase variant. In some preferred embodiments, the composition is enriched in the cellulase variant.

In other aspects, the present disclosure provides an isolated nucleic acid encoding the cellulase variant of any of the preceding paragraphs of the summary. An expression vector comprising the isolated nucleic acid operably linked to a regulatory sequence is also provided, as is a host cell comprising the expression vector. In some embodiments, methods are provided for producing a cellulase variant, comprising culturing the host cells in a culture medium under suitable conditions to produce the cellulase variant.

Also provided are compositions comprising the cellulase variant of any of the preceding paragraphs of the summary. In some preferred embodiments, the compositions further comprising at least one additional enzyme selected from the group consisting of a subtilisin, a neutral metalloprotease, a lipase, a cutinase, an amylase, a carbohydrase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, and a peroxidase. The disclosure further provides methods of cleaning or fabric care comprising contacting a surface or an article comprising a fabric with the composition. Additionally, methods are provided for depilling and surface finishing a fabric comprising contacting a surface or an article comprising a fabric with the composition.

In some preferred embodiments, methods are provided for converting biomass to sugars comprising contacting the biomass with the cellulase variant of any of the preceding paragraphs of the summary. In some preferred embodiments methods are provided for producing a fuel comprising: contacting a biomass composition with an enzymatic composition comprising the cellulase variant of any of the preceding paragraphs to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel.

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the scope and spirit of the disclosure will become apparent to one skilled in the art from reading this detailed description.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an alignment of the amino acid sequences of the mature form of various cellulases: Hypocrea jecorina (also known as T. reesei) CBH2 (SEQ ID NO:3), Hypocrea koningii CBH2 (SEQ ID NO:4), Humicola insolens CBH2 (SEQ ID NO:5), Acremonium cellulolyticus CBH2 (SEQ ID NO:6), Agaricus bisporus CBH2 (SEQ ID NO:7), Fusarium osysporum EG (SEQ ID NO:8), Phanerochaete chrysosporium CBH2 (SEQ ID NO:9), Talaromyces emersonii CBH2 (SEQ ID NO:10), Thermobifida. fusca 6B/E3 CBH2 (SEQ ID NO:11), Thermobifida fusca 6A/E2 EG (SEQ ID NO:12), and Cellulomonas fimi CenA EG (SEQ ID NO:13).

FIG. 2A provides a graph of the relative frequency of observed over expected pretreated corn stover (PCS) assay winners as a product of charge change of the CBH2 SELs. Decreasing CBH2 charge results in a significantly higher frequency of PCS assay winners. FIG. 2B provides a graph of the relative frequency of observed over expected phosphoric acid swollen cellulose (PASC) assay winners as a product of charge change of the CBH2 SELs. A significantly higher frequency of PASC winners is observed for variants without a charge change.

FIG. 3 provides a plasmid map for pTTTpyrG-cbh2.

FIG. 4 provides a schematic of the T. reesei ku80 deletion cassette.

FIG. 5 illustrates the inactivation of the native ku80 gene as a consequence of its replacement with an als selectable marker.

FIG. 6 provides a schematic of the T. reesei pyr2 deletion cassette.

FIG. 7 illustrates the inactivation of the native pyr2 gene as a consequence of its replacement with a hygR selectable marker and an amdS fragment.

FIG. 8 provides a schematic of the T. reesei hygR deletion cassette

FIG. 9 illustrates the inactivation of the heterologous hygR gene as a consequence of its replacement with a pyr2 selectable marker.

FIG. 10 provides a schematic of the T. reesei bgl1 deletion cassette

FIG. 11 provides a schematic of the T. reesei egl3 deletion cassette

FIG. 12 provides a schematic of the T. reesei telomeric plasmid vector used for expression of cre recombinase.

FIG. 13 illustrates the inactivation of the pyr2 selectable marker and activation of the amdS selectable marker as a consequence of introduction of a polynucleotide of a gene of interest (GOI). In exemplary embodiments, the GOI encodes a CBH2 variant.

FIG. 14 provides a schematic illustrating the derivation of the MAD6 host strain, from the quad-deleted strain.

V. DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

The present disclosure relates to enzymes and in particular cellulase variants. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, methods of identifying additional useful cellulase variants and methods of using the compositions.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods described herein. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this application, the use of the singular includes the plural unless specifically stated otherwise. The use of “or” means “and/or” unless state otherwise. Likewise, the terms “comprise,” “comprising,” “comprises,” “include,” “including” and “includes” are not intended to be limiting. All patents and publications, including all amino acid and nucleotide sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms herein are more fully defined by reference to the specification as a whole.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxyl orientation, respectively. Practitioners are particularly directed to Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel F M et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1993, for definitions and terms of the art. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary.

I. Definitions

The terms below are more fully defined by reference to the specification as a whole.

The term “polypeptide” as used herein refers to a compound made up of a single chain of amino acid residues linked by peptide bonds. The term “protein” as used herein may be synonymous with the term “polypeptide”.

“Variant” means a protein which is derived from a precursor protein (e.g., the native protein) by addition of one or more amino acids to either or both the C- and N-terminal end, substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, or deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence. The preparation of a cellulase variant may be performed by any means know in the art. In preferred embodiments, a cellulase variant is prepared by modifying a DNA sequence which encodes for the native protein, transformation of the modified DNA sequence into a suitable host, and expression of the modified DNA sequence to form the variant enzyme. The variant cellulase of the disclosure includes peptides comprising altered amino acid sequences in comparison with a precursor enzyme amino acid sequence wherein the variant cellulase retains the characteristic cellulolytic nature of the precursor enzyme but which may have altered properties in some specific aspect. For example, a variant cellulase may have an increased pH optimum or increased temperature or oxidative stability or decreased affinity or binding to non-cellulosic materials but will retain its characteristic cellulolytic activity. It is contemplated that the variants according to the present disclosure may be derived from a DNA fragment encoding a cellulase variant wherein the functional activity of the expressed cellulase variant is retained. For example, a DNA fragment encoding a cellulase may further include a DNA sequence or portion thereof encoding a hinge or linker attached to the cellulase DNA sequence at either the 5′ or 3′ end wherein the functional activity of the encoded cellulase domain is retained. The terms variant and derivative may be used interchangeably herein.

“Equivalent residues” may also be defined by determining homology at the level of tertiary structure for a precursor cellulase whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of a cellulase and Hypocrea jecorina CBH2 (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the cellulase in question to the H. jecorina CBH2. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available see for examples US 2006/0205042.

Equivalent residues which are functionally analogous to a specific residue of H. jecorina CBH2 are defined as those amino acids of a cellulase which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the H. jecorina CBH2. Further, they are those residues of the cellulase (for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of H. jecorina CBH2. The crystal structure of H. jecorina CBH2 is shown in Zou et al. (1999) (Ref. 5, supra).

The term “nucleic acid molecule” includes RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given protein such as CBH2 and/or variants thereof may be produced. The present disclosure contemplates every possible variant nucleotide sequence, encoding variant cellulase such as CBH2, all of which are possible given the degeneracy of the genetic code.

A “heterologous” nucleic acid construct or sequence has a portion of the sequence which is not native to the cell in which it is expressed. Heterologous, with respect to a control sequence refers to a control sequence (i.e. promoter or enhancer) that does not function in nature to regulate the same gene the expression of which it is currently regulating. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, transformation, microinjection, electroporation, or the like. A “heterologous” nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from a control sequence/DNA coding sequence combination found in the native cell.

As used herein, the term “vector” refers to a nucleic acid construct designed for transfer between different host cells. An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

Accordingly, an “expression cassette” or “expression vector” is a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes.

As used herein, the term “selectable marker-encoding nucleotide sequence” refers to a nucleotide sequence which is capable of expression in cells and where expression of the selectable marker confers to cells containing the expressed gene the ability to grow in the presence of a corresponding selective agent, or under corresponding selective growth conditions.

As used herein, the term “promoter” refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. The promoter will generally be appropriate to the host cell in which the target gene is being expressed. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences”) are necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

“Chimeric gene” or “heterologous nucleic acid construct”, as defined herein refers to a non-native gene (i.e., one that has been introduced into a host) that may be composed of parts of different genes, including regulatory elements. A chimeric gene construct for transformation of a host cell is typically composed of a transcriptional regulatory region (promoter) operably linked to a heterologous protein coding sequence, or, in a selectable marker chimeric gene, to a selectable marker gene encoding a protein conferring, for example, antibiotic resistance to transformed cells. A typical chimeric gene of the present disclosure, for transformation into a host cell, includes a transcriptional regulatory region that is constitutive or inducible, a protein coding sequence, and a terminator sequence. A chimeric gene construct may also include a second DNA sequence encoding a signal peptide if secretion of the target protein is desired.

A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors, linkers or primers for PCR are used in accordance with conventional practice.

As used herein, the term “gene” means the segment of DNA involved in producing a polypeptide chain, that may or may not include regions preceding and following the coding region, e.g. 5′ untranslated (5′ UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).

In general, nucleic acid molecules which encode the variant cellulase such as CBH2 will hybridize, under moderate to high stringency conditions to the wild type sequence such as provided herein as SEQ ID NO:1. However, in some cases a CBH2-encoding nucleotide sequence is employed that possesses a substantially different codon usage, while the protein encoded by the CBH2-encoding nucleotide sequence has the same or substantially the same amino acid sequence as the native protein. For example, the coding sequence may be modified to facilitate faster expression of CBH2 in a particular prokaryotic or eukaryotic expression system, in accordance with the frequency with which a particular codon is utilized by the host (Te'o et al., FEMS Microbiology Letters, 190: 13-19, 2000, for example, describes the optimization of genes for expression in filamentous fungi).

A nucleic acid sequence is considered to be “selectively hybridizable” to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm-5° C. (5° C. below the Tm of the probe); “high stringency” at about 5-10° C. below the Tm; “moderate” or “intermediate stringency” at about 10-20°. C. below the Tm of the probe; and “low stringency” at about 20-25° C. below the Tm. Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify sequences having about 80% or more sequence identity with the probe.

Moderate and high stringency hybridization conditions are well known in the art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, and in Ausubel, F. M., et al., 1993, expressly incorporated by reference herein). An example of high stringency conditions includes hybridization at about 42° C. in 50% formamide, 5×SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 ug/ml denatured carrier DNA followed by washing two times in 2.times.SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° degree. C.

The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

As used herein, the terms “transformed”, “stably transformed” or “transgenic” with reference to a cell means the cell has a non-native (heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.

As used herein, the term “expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).

It follows that the term “CBH2 expression” refers to transcription and translation of the cbh2 gene or variants thereof, the products of which include precursor RNA, mRNA, polypeptide, post-translationally processed polypeptides, and derivatives thereof, including CBH2 from related species such as Trichoderma koningii, Hypocrea jecorina (also known as Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride) and Hypocrea schweinitzii. By way of example, assays for CBH2 expression include Western blot for CBH2 protein, Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) assays for cbh2 mRNA, and Phosphoric Acid Swollen Cellulose (PASC) and p-hydroxybenzoic acid hydrazide (PAHBAH) assays as described in the following: (a) PASC: (Karlsson, J. et al. (2001), Eur. J. Biochem, 268, 6498-6507, Wood, T. (1988) in Methods in Enzymology, Vol. 160. Biomass Part a Cellulose and Hemicellulose (Wood, W. & Kellog, S. Eds.), pp. 19-25, Academic Press, San Diego, Calif., USA) and (b) PAHBAH: (Lever, M. (1972) Analytical Biochemistry, 47, 273, Blakeney, A. B. & Mutton, L. L. (1980) Journal of Science of Food and Agriculture, 31, 889, Henry, R. J. (1984) Journal of the Institute of Brewing, 90, 37).

The term “alternative splicing” refers to the process whereby multiple polypeptide isoforms are generated from a single gene, and involves the splicing together of nonconsecutive exons during the processing of some, but not all, transcripts of the gene. Thus a particular exon may be connected to any one of several alternative exons to form messenger RNAs. The alternatively-spliced mRNAs produce polypeptides (“splice variants”) in which some parts are common while other parts are different.

The term “signal sequence” refers to a sequence of amino acids at the N-terminal portion of a protein that facilitates the secretion of the mature form of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence that is cleaved off during the secretion process.

By the term “host cell” is meant a cell that contains a vector and supports the replication, and/or transcription or transcription and translation (expression) of the expression construct. Host cells for use in the present disclosure can be prokaryotic cells, such as E. coli, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells. In general, host cells are filamentous fungi.

The term “filamentous fungi” means any and all filamentous fungi recognized by those of skill in the art. A preferred fungus is selected from the group consisting of Aspergillus, Trichoderma, Fusarium, Chrysosporium, Penicillium, Humicola, Neurospora, or alternative sexual forms thereof such as Emericella, Hypocrea. It has now been demonstrated that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina (See, Kuhls et al., PNAS, 93:7755-7760, 1996).

The term “cellooligosaccharide” refers to oligosaccharide groups containing from 2-8 glucose units and having .beta.-1,4 linkages, e.g., cellobiose.

The terms “cellulase” “cellulolytic enzymes” or “cellulase enzymes” refer to a category of enzymes capable of hydrolyzing cellulose polymers to shorter cello-oligosaccharide oligomers, cellobiose and/or glucose. Numerous examples of cellulases, such as exoglucanases, exocellobiohydrolases, endoglucanases, and glucosidases have been obtained from cellulolytic organisms, particularly including fungi, plants and bacteria. The enzymes made by these microbes are mixtures of proteins with three types of actions useful in the conversion of cellulose to glucose: endoglucanases (EG), cellobiohydrolases (CBH), and beta-glucosidase. These three different types of cellulase enzymes act synergistically to convert cellulose and its derivatives to glucose.

Many microbes make enzymes that hydrolyze cellulose, including the wood rotting fungus Trichoderma, the compost bacteria Thermomonospora, Bacillus, and Cellulomonas; Streptomyces; and the fungi Humicola, Aspergillus and Fusarium.

CBH2 from Hypocrea jecorina is a member of the Glycosyl Hydrolase Family 6 (hence Cel6) and, specifically, was the first member of that family identified in Hypocrea jecorina (hence Cel6A). The Glycosyl Hydrolase Family 6 contains both Endoglucanases and Cellobiohydrolases/exoglucanases, and that CBH2 is the latter. Thus, the phrases CBH2, CBH2-type protein and Cel6 cellobiohydrolases may be used interchangeably herein.

The term “cellulose binding domain” as used herein refers to portion of the amino acid sequence of a cellulase or a region of the enzyme that is involved in the cellulose binding activity of a cellulase or derivative thereof. Cellulose binding domains generally function by non-covalently binding the cellulase to cellulose, a cellulose derivative or other polysaccharide equivalent thereof. Cellulose binding domains permit or facilitate hydrolysis of cellulose fibers by the structurally distinct catalytic core region, and typically function independent of the catalytic core. Thus, a cellulose binding domain will not possess the significant hydrolytic activity attributable to a catalytic core. In other words, a cellulose binding domain is a structural element of the cellulase enzyme protein tertiary structure that is distinct from the structural element which possesses catalytic activity. Cellulose binding domain and cellulose binding module may be used interchangeably herein.

As used herein, the term “surfactant” refers to any compound generally recognized in the art as having surface active qualities. Thus, for example, surfactants comprise anionic, cationic and nonionic surfactants such as those commonly found in detergents. Anionic surfactants include linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; and alkanesulfonates. Ampholytic surfactants include quaternary ammonium salt sulfonates, and betaine-type ampholytic surfactants. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule. Nonionic surfactants may comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like.

As used herein, the term “cellulose containing fabric” refers to any sewn or unsewn fabrics, yarns or fibers made of cotton or non-cotton containing cellulose or cotton or non-cotton containing cellulose blends including natural cellulosics and manmade cellulosics (such as jute, flax, ramie, rayon, and lyocell).

As used herein, the term “cotton-containing fabric” refers to sewn or unsewn fabrics, yarns or fibers made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, cotton yarns, raw cotton and the like.

As used herein, the term “stonewashing composition” refers to a formulation for use in stonewashing cellulose containing fabrics. Stonewashing compositions are used to modify cellulose containing fabrics prior to sale, i.e., during the manufacturing process. In contrast, detergent compositions are intended for the cleaning of soiled garments and are not used during the manufacturing process.

As used herein, the term “detergent composition” refers to a mixture which is intended for use in a wash medium for the laundering of soiled cellulose containing fabrics. In the context of the present disclosure, such compositions may include, in addition to cellulases and surfactants, additional hydrolytic enzymes, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, cellulase activators, antioxidants, and solubilizers.

As used herein, the term “decrease or elimination in expression of the cbh2 gene” means that either that the cbh2 gene has been deleted from the genome and therefore cannot be expressed by the recombinant host microorganism; or that the cbh2 gene or transcript has been modified such that a functional CBH2 enzyme is not produced by the host microorganism or at levels that are significantly less than the unmodified cbh2 gene or transcript.

The term “variant cbh2 gene” means that the nucleic acid sequence of the cbh2 gene from H. jecorina has been altered by removing, adding, and/or manipulating the coding sequence.

As used herein, the term “purifying” generally refers to subjecting transgenic nucleic acid or protein containing cells to biochemical purification and/or column chromatography.

As used herein, the terms “active” and “biologically active” refer to a biological activity associated with a particular protein and are used interchangeably herein. For example, the enzymatic activity associated with a protease is proteolysis and, thus, an active protease has proteolytic activity. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those of skill in the art.

As used herein, the term “enriched” means that the cellulase such as CBH2 is found in a concentration that is greater relative to the CBH2 concentration found in a wild-type, or naturally occurring, fungal cellulase composition. The terms enriched, elevated and enhanced may be used interchangeably herein.

A wild type fungal cellulase composition is one produced by a naturally occurring fungal source and which comprises one or more BGL, CBH and EG components wherein each of these components is found at the ratio produced by the fungal source. Thus, an enriched CBH composition would have CBH at an altered ratio wherein the ratio of CBH to other cellulase components (i.e., EGs, beta-glucosidases and other endoglucanases) is elevated. This ratio may be increased by either increasing CBH or decreasing (or eliminating) at least one other component by any means known in the art.

The term “isolated” or “purified” as used herein refers to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.

Thus, to illustrate, a naturally occurring cellulase system may be purified into substantially pure components by recognized separation techniques well published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion and the like. For example, in ion exchange chromatography (usually anion exchange chromatography), it is possible to separate the cellulase components by eluting with a pH gradient, or a salt gradient, or both a pH and a salt gradient. The purified CBH may then be added to the enzymatic solution resulting in an enriched CBH solution. It is also possible to elevate the amount of CBH produced by a microbe using molecular genetics methods to overexpress the gene encoding CBH, possibly in conjunction with deletion of one or more genes encoding other cellulases.

Fungal cellulases may contain more than one CBH component. The different components generally have different isoelectric points which allow for their separation via ion exchange chromatography and the like. Either a single CBH component or a combination of CBH components may be employed in an enzymatic solution.

When employed in enzymatic solutions, the homolog or variant CBH2 component is generally added in an amount sufficient to allow the highest rate of release of soluble sugars from the biomass. The amount of homolog or variant CBH2 component added depends, upon the type of biomass to be saccharified, which can be readily determined by the skilled artisan when employed, the weight percent of the homolog or variant CBH2 component present in the cellulase composition is from preferably between 1 and 100 with illustrative examples being about 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Furthermore, preferred ranges may be about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 60 weight percent, from about 15 to about 65 weight percent, from about 15 to about 70 weight percent, from about 15 to about 75 weight percent, from about 15 to about 80 weight percent, from about 15 to about 85 weight percent, from about 15 to about 95 weight percent. However, when employed, the weight percent of the homolog or variant CBH2 component relative to any EG type components present in the cellulase composition is from preferably about 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Furthermore, preferred ranges may be about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 20 weight percent, from about 15 to about 25 weight percent, from about 15 to about 30 weight percent, from about 15 to about 35 weight percent, from about 15 to about 30 weight percent, from about 15 to about 45 weight percent, from about 15 to about 50 weight percent.

II. Cellulases

Cellulases are known in the art as enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. As set forth above, cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91) (“CBH”) and beta-glucosidases (EC 3.2.1.21) (“BG”).

Certain fungi produce complete cellulase systems which include exo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-type cellulases and beta-glucosidases or BG-type cellulases. However, sometimes these systems lack CBH-type cellulases and bacterial cellulases also typically include little or no CBH-type cellulases. In addition, it has been shown that the EG components and CBH components synergistically interact to more efficiently degrade cellulose. The different components, i.e., the various endoglucanases and exocellobiohydrolases in a multi-component or complete cellulase system, generally have different properties, such as isoelectric point, molecular weight, degree of glycosylation, substrate specificity and enzymatic action patterns.

It is believed that endoglucanase-type cellulases hydrolyze internal beta-1,4-glucosidic bonds in regions of low crystallinity of the cellulose and exo-cellobiohydrolase-type cellulases hydrolyze cellobiose from the reducing or non-reducing end of cellulose. It follows that the action of endoglucanase components can greatly facilitate the action of exo-cellobiohydrolases by creating new chain ends which are recognized by exo-cellobiohydrolase components. Further, beta-glucosidase-type cellulases have been shown to catalyze the hydrolysis of alkyl and/or aryl .beta.-D-glucosides such as methyl .beta.-D-glucoside and p-nitrophenyl glucoside as well as glycosides containing only carbohydrate residues, such as cellobiose. This yields glucose as the sole product for the microorganism and reduces or eliminates cellobiose which inhibits cellobiohydrolases and endoglucanases.

Cellulases also find a number of uses in detergent compositions including to enhance cleaning ability, as a softening agent and to improve the feel of cotton fabrics (Hemmpel, ITB Dyeing/Printing/Finishing 3:5-14, 1991; Tyndall, Textile Chemist and Colorist 24:23-26, 1992; and Kumar et al., Textile Chemist and Colorist, 29:37-42, 1997). While the mechanism is not part of the disclosure, softening and color restoration properties of cellulase have been attributed to the alkaline endoglucanase components in cellulase compositions, as exemplified by U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757, which disclose that detergent compositions containing a cellulase composition enriched in a specified alkaline endoglucanase component impart color restoration and improved softening to treated garments as compared to cellulase compositions not enriched in such a component. In addition, the use of such alkaline endoglucanase components in detergent compositions has been shown to complement the pH requirements of the detergent composition (e.g., by exhibiting maximal activity at an alkaline pH of 7.5 to 10, as described in U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757).

Cellulase compositions have also been shown to degrade cotton-containing fabrics, resulting in reduced strength loss in the fabric (U.S. Pat. No. 4,822,516), contributing to reluctance to use cellulase compositions in commercial detergent applications. Cellulase compositions comprising endoglucanase components have been suggested to exhibit reduced strength loss for cotton-containing fabrics as compared to compositions comprising a complete cellulase system.

Cellulases have also been shown to be useful in degradation of cellulase biomass to ethanol (wherein the cellulase degrades cellulose to glucose and yeast or other microbes further ferment the glucose into ethanol), in the treatment of mechanical pulp (Pere et al., In Proc. Tappi Pulping Conf., Nashville, Term., 27-31, pp. 693-696, 1996), for use as a feed additive (WO 91/04673) and in grain wet milling

Most CBHs and EGs have a multidomain structure consisting of a core domain separated from a cellulose binding domain (CBD) by a linker peptide (Suurnakki et al., 2000). The core domain contains the active site whereas the CBD interacts with cellulose by binding the enzyme to it (van Tilbeurgh et al., FEBS Lett. 204:223-227, 1986; Tomme et al., Eur. J. Biochem. 170:575-581, 1988). The CBDs are particularly important in the hydrolysis of crystalline cellulose. It has been shown that the ability of cellobiohydrolases to degrade crystalline cellulose clearly decreases when the CBD is absent (Linder and Teeri, J. Biotechnol. 57:15-28, 1997). However, the exact role and action mechanism of CBDs is still a matter of speculation. It has been suggested that the CBD enhances the enzymatic activity merely by increasing the effective enzyme concentration at the surface of cellulose (Stahlberg et al., Bio/Technol. 9:286-290, 1991), and/or by loosening single cellulose chains from the cellulose surface (Tormo et al., EMBO J. vol. 15, no. 21, pp. 5739-5751, 1996). Most studies concerning the effects of cellulase domains on different substrates have been carried out with core proteins of cellobiohydrolases, as their core proteins can easily be produced by limited proteolysis with papain (Tomme et al., 1988). Numerous cellulases have been described in the scientific literature, examples of which include: from Trichoderma reesei: Shoemaker, S. et al., Bio/Technology, 1:691-696, 1983, which discloses CBH1; Teeri, T. et al., Gene, 51:43-52, 1987, which discloses CBH2. Cellulases from species other than Trichoderma have also been described e.g., Ooi et al., Nucleic Acids Research, vol. 18, no. 19, 1990, which discloses the cDNA sequence coding for endoglucanase F1-CMC produced by Aspergillus aculeatus; Kawaguchi T et al., Gene 173 (2):287-8, 1996, which discloses the cloning and sequencing of the cDNA encoding beta-glucosidase 1 from Aspergillus aculeatus; Sakamoto et al., Curr. Genet. 27:435-439, 1995, which discloses the cDNA sequence encoding the endoglucanase CMCase-1 from Aspergillus kawachii IFO 4308; Saarilahti et al., Gene 90:9-14, 1990, which discloses an endoglucanase from Erwinia carotovara; Spilliaert R, et al., Eur J. Biochem. 224 (3):923-30, 1994, which discloses the cloning and sequencing of bglA, coding for a thermostable beta-glucanase from Rhodothermus marinus; and Halldorsdottir S et al., Appl Microbiol Biotechnol. 49 (3):277-84, 1998, which discloses the cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. However, there remains a need for identification and characterization of novel cellulases, with improved properties, such as improved performance under conditions of thermal stress or in the presence of surfactants, increased specific activity, altered substrate cleavage pattern, and/or high level expression in vitro.

The development of new and improved cellulase compositions that comprise varying amounts CBH-type, EG-type and BG-type cellulases is of interest for use: (1) in detergent compositions that exhibit enhanced cleaning ability, function as a softening agent and/or improve the feel of cotton fabrics (e.g., “stone washing” or “biopolishing”); (2) in compositions for degrading wood pulp or other biomass into sugars (e.g., for bio-fuel production); and/or (3) in feed compositions.

Also provided herein are whole cellulase preparation comprising cellulase variants. As used herein, the phrase “whole cellulase preparation” refers to both naturally occurring and non-naturally occurring cellulase containing compositions. A “naturally occurring” composition is one produced by a naturally occurring source and which comprises one or more cellobiohydrolase-type, one or more endoglucanase-type, and one or more beta-glucosidase components wherein each of these components is found at the ratio produced by the source. A naturally occurring composition is one that is produced by an organism unmodified with respect to the cellulolytic enzymes such that the ratio of the component enzymes is unaltered from that produced by the native organism. A “non-naturally occurring” composition encompasses those compositions produced by: (1) combining component cellulolytic enzymes either in a naturally occurring ratio or non-naturally occurring, i.e., altered, ratio; or (2) modifying an organism to overexpress or underexpress one or more cellulolytic enzyme; or (3) modifying an organism such that at least one cellulolytic enzyme is deleted. Accordingly, in some embodiments, the whole cellulase preparation can have one or more of the various EGs and/or CBHs, and/or beta-glucosidase deleted. For example, EG1 may be deleted alone or in combination with other EGs and/or CBHs.

In general, the whole cellulase preparation includes enzymes including, but are not limited to: (i) endoglucanases (EG) or 1,4-β-d-glucan-4-glucanohydrolases (EC 3.2.1.4), (ii) exoglucanases, including 1,4-β-d-glucan glucanohydrolases (also known as cellodextrinases) (EC 3.2.1.74) and 1,4-β-d-glucan cellobiohydrolases (exo-cellobiohydrolases, CBH) (EC 3.2.1.91), and (iii) β-glucosidase (BG) or β-glucoside glucohydrolases (EC 3.2.1.21).

In the present disclosure, the whole cellulase preparation can be from any microorganism that is useful for the hydrolysis of a cellulosic material. In some embodiments, the whole cellulase preparation is a filamentous fungi whole cellulase. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota. In some embodiments, the whole cellulase preparation is a Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Scytalidium, Thielavia, Tolypocladium, or Trichoderma species, whole cellulase. In some embodiments, the whole cellulase preparation is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae whole cellulase. In another aspect, whole cellulase preparation is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum whole cellulase. In another aspect, the whole cellulase preparation is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Scytalidium thermophilum, or Thielavia terrestris whole cellulase. In another aspect, the whole cellulase preparation a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei e.g., RL-P37 (Sheir-Neiss et al., Appl. Microbiol. Biotechnology, 20 (1984) pp. 46-53; Montenecourt B. S., Can., 1-20, 1987), QM9414 (ATCC No. 26921), NRRL 15709, ATCC 13631, 56764, 56466, 56767, or Trichoderma viride e.g., ATCC 32098 and 32086, whole cellulase. In some embodiments, the whole cellulase preparation is a Trichoderma reesei RutC30 whole cellulase, which is available from the American Type Culture Collection as Trichoderma reesei ATCC 56765.

Examples of commercial cellulase preparations suitable for use in the present disclosure include, for example, CELLUCLAST™ (available from Novozymes A/S) and LAMINEX™, IndiAge™ and Primafast™ LAMINEX BG enzyme (available Genencor Division, Danisco US. Inc.)

In the present disclosure, the whole cellulase preparation can be from any microorganism cultivation method known in the art resulting in the expression of enzymes capable of hydrolyzing a cellulosic material. Fermentation can include shake flask cultivation, small- or large-scale fermentation, such as continuous, batch, fed-batch, or solid state fermentations in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the cellulase to be expressed or isolated.

Generally, the microorganism is cultivated in a cell culture medium suitable for production of enzymes capable of hydrolyzing a cellulosic material. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable culture media, temperature ranges and other conditions suitable for growth and cellulase production are known in the art. As a non-limiting example, the normal temperature range for the production of cellulases by Trichoderma reesei is 24° C. to 28° C.

Generally, the whole cellulase preparation is used as is produced by fermentation with no or minimal recovery and/or purification. For example, once cellulases are secreted by a cell into the cell culture medium, the cell culture medium containing the cellulases can be used. In some embodiments the whole cellulase preparation comprises the unfractionated contents of fermentation material, including cell culture medium, extracellular enzymes and cells. Alternatively, the whole cellulase preparation can be processed by any convenient method, e.g., by precipitation, centrifugation, affinity, filtration or any other method known in the art. In some embodiments, the whole cellulase preparation can be concentrated, for example, and then used without further purification. In some embodiments the whole cellulase preparation comprises chemical agents that decrease cell viability or kills the cells. In some embodiments, the cells are lysed or permeabilized using methods known in the art.

III. Molecular Biology

In one embodiment this disclosure provides for the expression of variant cbh2 genes under control of a promoter functional in a filamentous fungus. Therefore, this disclosure relies on routine techniques in the field of recombinant genetics (See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, New York, 1994).

Methods of Mutating cbh2 Nucleic Acid Sequences

Any method known in the art that can introduce mutations is contemplated by the present disclosure.

The present disclosure relates to the expression, purification and/or isolation and use of variant CBH2. These enzymes are preferably prepared by recombinant methods utilizing the cbh2 gene from H. jecorina. The fermentation broth may be used with or without purification.

After the isolation and cloning of the cbh2 gene from H. jecorina, other methods known in the art, such as site directed mutagenesis, are used to make the substitutions, additions or deletions that correspond to substituted amino acids in the expressed CBH2 variant. Again, site directed mutagenesis and other methods of incorporating amino acid changes in expressed proteins at the DNA level are known in the art (Sambrook et al., supra; and Ausubel et al., supra).

DNA encoding an amino acid sequence variant of the H. jecorina CBH2 is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the H. jecorina CBH2.

Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (see, e.g., Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488 (1987)). Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide, i.e., H. jecorina CBH2. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). See, also, for example Cadwell et al., PCR Methods and Applications, Vol 2, 28-33 (1992). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34:315-323 (1985). The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5′ and 3′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence.

Alternatively, or additionally, the desired amino acid sequence encoding a variant CBH2 can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically.

The variant CBH2(s) so prepared may be subjected to further modifications, oftentimes depending on the intended use of the cellulase. Such modifications may involve further alteration of the amino acid sequence, fusion to heterologous polypeptide(s) and/or covalent modifications.

IV. cbh2 Nucleic Acids and CBH2 Polypeptides

A. Variant cbh2-Type Nucleic Acids

The nucleic acid sequence for the wild type cbh2 is shown in SEQ ID NO:1. The disclosure encompasses a nucleic acid molecule encoding the variant cellulases described herein. The nucleic acid may be a DNA molecule.

After DNA sequences that encode the CBH2 variants have been cloned into DNA constructs, the DNA is used to transform microorganisms. The microorganism to be transformed for the purpose of expressing a variant CBH2 according to the present disclosure may advantageously comprise a strain derived from Trichoderma sp. Thus, a preferred mode for preparing variant CBH2 cellulases according to the present disclosure comprises transforming a Trichoderma sp. host cell with a DNA construct comprising at least a fragment of DNA encoding a portion or all of the variant CBH2. The DNA construct will generally be functionally attached to a promoter. The transformed host cell is then grown under conditions so as to express the desired protein. Subsequently, the desired protein product may be purified to substantial homogeneity.

However, it may in fact be that the best expression vehicle for a given DNA encoding a variant CBH2 may differ from H. jecorina. Thus, it may be that it will be most advantageous to express a protein in a transformation host that bears phylogenetic similarity to the source organism for the variant CBH2. In an alternative embodiment, Aspergillus niger can be used as an expression vehicle. For a description of transformation techniques with A. niger, see WO 98/31821, the disclosure of which is incorporated by reference in its entirety.

Accordingly, the present description of an Aspergillus spp. expression system is provided for illustrative purposes only and as one option for expressing the variant CBH2 of the disclosure. One of skill in the art, however, may be inclined to express the DNA encoding variant CBH2 in a different host cell if appropriate and it should be understood that the source of the variant CBH2 should be considered in determining the optimal expression host. Additionally, the skilled worker in the field will be capable of selecting the best expression system for a particular gene through routine techniques utilizing the tools available in the art.

B. Variant CBH2 Polypeptides

The variant CBH2's of this disclosure have amino acid sequences that are derived from the amino acid sequence of a precursor CBH2. The amino acid sequence of the CBH2 variant differs from the precursor CBH2 amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. In a preferred embodiment, the precursor CBH2 is Hypocrea jecorina CBH2. The mature amino acid sequence of H. jecorina CBH2 is shown in Example 2 (SEQ ID NO:3). Thus, this disclosure is directed to CBH2 variants which contain amino acid residues at positions which are equivalent to the particular identified residue in H. jecorina CBH2. A residue (amino acid) of an CBH2 homolog is equivalent to a residue of Hypocrea jecorina CBH2 if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or is functionally analogous to a specific residue or portion of that residue in Hypocrea jecorina CBH2 (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally). As used herein, numbering is intended to correspond to that of the mature CBH2 amino acid sequence (SEQ ID NO:3).

Alignment of amino acid sequences to determine homology is preferably determined by using a “sequence comparison algorithm.” Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection, Visual inspection may utilize graphics packages such as, for example, MOE by Chemical Computing Group, Montreal Canada.

An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparison of both strands.

The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a protease if the smallest sum probability in a comparison of the test amino acid sequence to a protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

For purposes of the present disclosure, the degree of identity may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for polynucleotide sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

A structural alignment between a T. reesei CBH2 and other cellulases may be used to identify equivalent/corresponding positions in other cellulases having a moderate to high degree of homology, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%, with T. reesei CBH2 (SEQ ID NO: 3). One method of obtaining the structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., FEBS Letters, 224:149-155, 1987) and reverse threading (Huber and Torda, Protein Science, 7:142-149, 1998).

An exemplary alignment of the mature form of various reference cellulases is provided as FIG. 1. The reference cellulases include: Hypocrea jecorina (also known as T. reesei) CBH2 (SEQ ID NO:3), Hypocrea koningii CBH2 (SEQ ID NO:4), Humicola insolens CBH2 (SEQ ID NO:5), Acremonium cellulolyticus CBH2 (SEQ ID NO:6), Agaricus bisporus CBH2 (SEQ ID NO:7), Fusarium osysporum EG (SEQ ID NO:8), Phanerochaete chrysosporium CBH2 (SEQ ID NO:9), Talaromyces emersonii CBH2 (SEQ ID NO:10), Thermobifida. fusca 6B/E3 CBH2 (SEQ ID NO:11), Thermobifida fusca 6A/E2 EG (SEQ ID NO:12), and Cellulomonas fimi CenA EG (SEQ ID NO:13). Sequences were aligned using the ClustalW and MUSCLE multiple sequence alignment algorithms. A matrix showing the percent identity of cellulases of the sequence alignment of FIG. 1 is provided in Table 1.

TABLE 1 Cellulase Percent Identity Matrix* Percent_ID 3 4 5 6 7 8 9 10 11 12 13 3 100 95.5 62.3 64.7 59.6 63.1 55.4 63.4 31.9 13.5 27 4 95.5 100 61.6 64 59.1 63.6 54.7 63 32.9 13.5 26.8 5 62.3 61.6 100 59.1 57.6 61.3 54 58.8 31.9 15.9 26.6 6 64.7 64 59.1 100 58.6 56.4 54 72.6 32.8 13.5 29.2 7 59.6 59.1 57.6 58.6 100 55.8 69.1 58.1 34.9 17.5 27.6 8 63.1 63.6 61.3 56.4 55.8 100 48.7 54.8 31.1 13.9 25.2 9 55.4 54.7 54 54 69.1 48.7 100 52.6 32.4 15.4 25.6 10 63.4 63 58.8 72.6 58.1 54.8 52.6 100 33.9 13.2 27.3 11 31.9 32.9 31.9 32.8 34.9 31.1 32.4 33.9 100 15.9 36.3 12 13.5 13.5 15.9 13.5 17.5 13.9 15.4 13.2 15.9 100 12.8 13 27 26.8 26.6 29.2 27.6 25.2 25.6 27.3 36.3 12.8 100 *Numbers in the top row and left column correspond to the SEQ ID NOS of the aligned sequences of FIG. 1.

Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, et al., 1997.)

V. Expression of Recombinant CBH2 Variants

The methods of the disclosure rely on the use cells to express variant CBH2, with no particular method of CBH2 expression required. The variant CBH2 is preferably secreted from the cells. The disclosure provides host cells which have been transduced, transformed or transfected with an expression vector comprising a variant CBH2-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the parental host cell prior to transduction, transformation or transfection and will be apparent to those skilled in the art.

In one approach, a filamentous fungal cell or yeast cell is transfected with an expression vector having a promoter or biologically active promoter fragment or one or more (e.g., a series) of enhancers which functions in the host cell line, operably linked to a DNA segment encoding variant CBH2, such that variant CBH2 is expressed in the cell line.

A. Nucleic Acid Constructs/Expression Vectors.

Natural or synthetic polynucleotide fragments encoding variant CBH2 (“CBH2-encoding nucleic acid sequences”) may be incorporated into heterologous nucleic acid constructs or vectors, capable of introduction into, and replication in, a filamentous fungal or yeast cell. The vectors and methods disclosed herein are suitable for use in host cells for the expression of variant CBH2. Any vector may be used as long as it is replicable and viable in the cells into which it is introduced. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Cloning and expression vectors are also described in Sambrook et al., 1989, Ausubel F M et al., 1989, and Strathern et al., The Molecular Biology of the Yeast Saccharomyces, 1981, each of which is expressly incorporated by reference herein. Appropriate expression vectors for fungi are described in van den Hondel, C. A. M. J. J. et al. (1991) In: Bennett, J. W. and Lasure, L. L. (eds.) More Gene Manipulations in Fungi. Academic Press, pp. 396-428. The appropriate DNA sequence may be inserted into a plasmid or vector (collectively referred to herein as “vectors”) by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by standard procedures. Such procedures and related sub-cloning procedures are deemed to be within the scope of knowledge of those skilled in the art.

Recombinant filamentous fungi comprising the coding sequence for variant CBH2 may be produced by introducing a heterologous nucleic acid construct comprising the variant CBH2 coding sequence into the cells of a selected strain of the filamentous fungi.

Once the desired form of a variant cbh2 nucleic acid sequence is obtained, it may be modified in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions, and insertions may be performed on the naturally occurring sequence.

A selected variant cbh2 coding sequence may be inserted into a suitable vector according to well-known recombinant techniques and used to transform filamentous fungi capable of CBH2 expression. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express variant CBH2. Therefore it is appreciated that such substitutions in the coding region fall within the sequence variants covered by the present disclosure. Any and all of these sequence variants can be utilized in the same way as described herein for a parent CBH2-encoding nucleic acid sequence.

The present disclosure also includes recombinant nucleic acid constructs comprising one or more of the variant CBH2-encoding nucleic acid sequences as described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the disclosure has been inserted, in a forward or reverse orientation.

Heterologous nucleic acid constructs may include the coding sequence for variant cbh2. (i) in isolation; (ii) in combination with additional coding sequences; such as fusion protein or signal peptide coding sequences, where the cbh2 coding sequence is the dominant coding sequence; (iii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5′ and/or 3′ untranslated regions, effective for expression of the coding sequence in a suitable host; and/or (iv) in a vector or host environment in which the cbh2 coding sequence is a heterologous gene.

In one aspect of the present disclosure, a heterologous nucleic acid construct is employed to transfer a variant CBH2-encoding nucleic acid sequence into a cell in vitro, with established filamentous fungal and yeast lines preferred. For long-term, production of variant CBH2, stable expression is preferred. It follows that any method effective to generate stable transformants may be used in practicing the disclosure.

Appropriate vectors are typically equipped with a selectable marker-encoding nucleic acid sequence, insertion sites, and suitable control elements, such as promoter and termination sequences. The vector may comprise regulatory sequences, including, for example, non-coding sequences, such as introns and control elements, i.e., promoter and terminator elements or 5′ and/or 3′ untranslated regions, effective for expression of the coding sequence in host cells (and/or in a vector or host cell environment in which a modified soluble protein antigen coding sequence is not normally expressed), operably linked to the coding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, many of which are commercially available and/or are described in Sambrook, et al., (supra).

Exemplary promoters include both constitutive promoters and inducible promoters, examples of which include a CMV promoter, an SV40 early promoter, an RSV promoter, an EF-1.alpha. promoter, a promoter containing the tet responsive element (TRE) in the tet-on or tet-off system as described (ClonTech and BASF), the beta actin promoter and the metallothionine promoter that can upregulated by addition of certain metal salts. A promoter sequence is a DNA sequence which is recognized by the particular filamentous fungus for expression purposes. It is operably linked to DNA sequence encoding a variant CBH2 polypeptide. Such linkage comprises positioning of the promoter with respect to the initiation codon of the DNA sequence encoding the variant CBH2 polypeptide in the disclosed expression vectors. The promoter sequence contains transcription and translation control sequence which mediate the expression of the variant CBH2 polypeptide. Examples include the promoters from the Aspergillus niger, A. awamori or A. oryzae glucoamylase, alpha-amylase, or alpha-glucosidase encoding genes; the A. nidulans gpdA or trpC Genes; the Neurospora crassa cbh1 or trp1 genes; the A. niger or Rhizomucor miehei aspartic proteinase encoding genes; the H. jecorina (T. reesei) cbh1, cbh2, egl1, egl2, or other cellulase encoding genes.

The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art. Typical selectable marker genes include argB from A. nidulans or T. reesei, amdS from A. nidulans, pyr4 from Neurospora crassa or T. reesei, pyrG from Aspergillus niger or A. nidulans. Additional exemplary selectable markers include, but are not limited to trpc, trp1, oliC31, niaD or leu2, which are included in heterologous nucleic acid constructs used to transform a mutant strain such as trp-, pyr-, leu- and the like.

Such selectable markers confer to transformants the ability to utilize a metabolite that is usually not metabolized by the filamentous fungi. For example, the amdS gene from H. jecorina which encodes the enzyme acetamidase that allows transformant cells to grow on acetamide as a nitrogen source. The selectable marker (e.g. pyrG) may restore the ability of an auxotrophic mutant strain to grow on a selective minimal medium or the selectable marker (e.g. olic31) may confer to transformants the ability to grow in the presence of an inhibitory drug or antibiotic.

The selectable marker coding sequence is cloned into any suitable plasmid using methods generally employed in the art. Exemplary plasmids include pUC18, pBR322, pRAX and pUC100. The pRAX plasmid contains AMAL sequences from A. nidulans, which make it possible to replicate in A. niger.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., 1989; Freshney, Animal Cell Culture, 1987; Ausubel, et al., 1993; and Coligan et al., Current Protocols in Immunology, 1991.

B. Host Cells and Culture Conditions for CBH2 Production

(i) Filamentous Fungi

Thus, the present disclosure provides filamentous fungi comprising cells which have been modified, selected and cultured in a manner effective to result in variant CBH2 production or expression relative to the corresponding non-transformed parental fungi.

Examples of species of parental filamentous fungi that may be treated and/or modified for variant CBH2 expression include, but are not limited to Trichoderma, e.g., Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii; Penicillium sp., Humicola sp., including Humicola insolens; Aspergillus sp., Chrysosporium sp., Fusarium sp., Hypocrea sp., and Emericella sp.

CBH2 expressing cells are cultured under conditions typically employed to culture the parental fungal line. Generally, cells are cultured in a standard medium containing physiological salts and nutrients, such as described in Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, J. P. et al., Academic Press, pp. 71-86, 1988 and Ilmen, M. et al., Appl. Environ. Microbiol. 63:1298-1306, 1997. Culture conditions are also standard, e.g., cultures are incubated at 28.degree. C. in shaker cultures or fermenters until desired levels of CBH2 expression are achieved.

Preferred culture conditions for a given filamentous fungus may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection (ATCC; www.atcc.org/). After fungal growth has been established, the cells are exposed to conditions effective to cause or permit the expression of variant CBH2.

In cases where a CBH2 coding sequence is under the control of an inducible promoter, the inducing agent, e.g., a sugar, metal salt or antibiotics, is added to the medium at a concentration effective to induce CBH2 expression.

In one embodiment, the strain comprises Aspergillus niger, which is a useful strain for obtaining overexpressed protein. For example A. niger var awamori dgr246 is known to secrete elevated amounts of secreted cellulases (Goedegebuur et al., Curr. Genet (2002) 41: 89-98). Other strains of Aspergillus niger var awamori such as GCDAP3, GCDAP4 and GAP3-4 are known Ward et al (Ward, M, Wilson, L. J. and Kodama, K. H., 1993, Appl. Microbiol. Biotechnol. 39:738-743).

In another embodiment, the strain comprises Trichoderma reesei, which is a useful strain for obtaining overexpressed protein. For example, RL-P37, described by Sheir-Neiss, et al., Appl. Microbiol. Biotechnol. 20:46-53 (1984) is known to secrete elevated amounts of cellulase enzymes. Functional equivalents of RL-P37 include Trichoderma reesei strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). It is contemplated that these strains would also be useful in overexpressing variant CBH2.

Where it is desired to obtain the variant CBH2 in the absence of potentially detrimental native cellulolytic activity, it is useful to obtain a Trichoderma host cell strain which has had one or more cellulase genes deleted prior to introduction of a DNA construct or plasmid containing the DNA fragment encoding the variant CBH2. Such strains may be prepared by the method disclosed in U.S. Pat. No. 5,246,853 and WO 92/06209, which disclosures are hereby incorporated by reference. By expressing a variant CBH2 cellulase in a host microorganism that is missing one or more cellulase genes, the identification and subsequent purification procedures are simplified. Any gene from Trichoderma sp. which has been cloned can be deleted, for example, the cbh1, cbh2, egl1, and egl2 genes as well as those encoding EG III and/or EGV protein (see e.g., U.S. Pat. No. 5,475,101 and WO 94/28117, respectively).

Gene deletion may be accomplished by inserting a form of the desired gene to be deleted or disrupted into a plasmid by methods known in the art. The deletion plasmid is then cut at an appropriate restriction enzyme site(s), internal to the desired gene coding region, and the gene coding sequence or part thereof replaced with a selectable marker. Flanking DNA sequences from the locus of the gene to be deleted or disrupted, preferably between about 0.5 to 2.0 kb, remain on either side of the selectable marker gene. An appropriate deletion plasmid will generally have unique restriction enzyme sites present therein to enable the fragment containing the deleted gene, including flanking DNA sequences, and the selectable marker gene to be removed as a single linear piece.

A selectable marker must be chosen so as to enable detection of the transformed microorganism. Any selectable marker gene that is expressed in the selected microorganism will be suitable. For example, with Aspergillus sp., the selectable marker is chosen so that the presence of the selectable marker in the transformants will not significantly affect the properties thereof. Such a selectable marker may be a gene that encodes an assayable product. For example, a functional copy of an Aspergillus sp. gene may be used which if lacking in the host strain results in the host strain displaying an auxotrophic phenotype. Similarly, selectable markers exist for Trichoderma sp.

In one embodiment, a pyrG-derivative strain of Aspergillus sp. is transformed with a functional pyrG gene, which thus provides a selectable marker for transformation. A pyrG-derivative strain may be obtained by selection of Aspergillus sp. strains that are resistant to fluoroorotic acid (FOA). The pyrG gene encodes orotidine-5′-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyrG gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyrG-derivative strains that lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique it is also possible to obtain uridine-requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy of the gene encoding this enzyme (Berges & Barreau, Curr. Genet. 19:359-365 (1991), and van Hartingsveldt et al., (1986) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol. Gen. Genet. 206:71-75). Selection of derivative strains is easily performed using the FOA resistance technique referred to above, and thus, the pyrG gene is preferably employed as a selectable marker.

In a second embodiment, a pyr4.-derivative strain of Hyprocrea sp. (Hyprocrea sp. (Trichoderma sp.)) is transformed with a functional pyr4 gene, which thus provides a selectable marker for transformation. A pyr4.sup.-derivative strain may be obtained by selection of Hyprocrea sp. (Trichoderma sp.) strains that are resistant to fluoroorotic acid (FOA). The pyr4 gene encodes orotidine-5′-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyr4 gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyr4.sup.-derivative strains that lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique it is also possible to obtain uridine-requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy of the gene encoding this enzyme (Berges & Barreau, 1991). Selection of derivative strains is easily performed using the FOA resistance technique referred to above, and thus, the pyr4 gene is preferably employed as a selectable marker.

To transform pyrG.-Aspergillus sp. or pyr4-Hyprocrea sp. (Trichoderma sp.) so as to be lacking in the ability to express one or more cellulase genes, a single DNA fragment comprising a disrupted or deleted cellulase gene is then isolated from the deletion plasmid and used to transform an appropriate pyr-Aspergillus or pyr-Trichoderma host. Transformants are then identified and selected based on their ability to express the pyrG or pyr4, respectively, gene product and thus compliment the uridine auxotrophy of the host strain. Southern blot analysis is then carried out on the resultant transformants to identify and confirm a double crossover integration event that replaces part or all of the coding region of the genomic copy of the gene to be deleted with the appropriate pyr selectable markers.

Although the specific plasmid vectors described above relate to preparation of pyr-transformants, the present disclosure is not limited to these vectors. Various genes can be deleted and replaced in the Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.) strain using the above techniques. In addition, any available selectable markers can be used, as discussed above. In fact, any host, e.g., Aspergillus sp. or Hyprocrea sp., gene that has been cloned, and thus identified, can be deleted from the genome using the above-described strategy.

As stated above, the host strains used may be derivatives of Hyprocrea sp. (Trichoderma sp.) that lack or have a nonfunctional gene or genes corresponding to the selectable marker chosen. For example, if the selectable marker of pyrG is chosen for Aspergillus sp., then a specific pyrG-derivative strain is used as a recipient in the transformation procedure. Also, for example, if the selectable marker of pyr4 is chosen for a Hyprocrea sp., then a specific pyr4-derivative strain is used as a recipient in the transformation procedure. Similarly, selectable markers comprising Hyprocrea sp. (Trichoderma sp.) genes equivalent to the Aspergillus nidulans genes amdS, argB, trpC, niaD may be used. The corresponding recipient strain must therefore be a derivative strain such as argB-, trpC-, niaD-, respectively.

DNA encoding the CBH2 variant is then prepared for insertion into an appropriate microorganism. According to the present disclosure, DNA encoding a CBH2 variant comprises the DNA necessary to encode for a protein that has functional cellulolytic activity. The DNA fragment encoding the CBH2 variant may be functionally attached to a fungal promoter sequence, for example, the promoter of the glaA gene in Aspergillus or the promoter of the cbh1 or egl1 genes in Trichoderma.

It is also contemplated that more than one copy of DNA encoding a CBH2 variant may be recombined into the strain to facilitate overexpression. The DNA encoding the CBH2 variant may be prepared by the construction of an expression vector carrying the DNA encoding the variant. The expression vector carrying the inserted DNA fragment encoding the CBH2 variant may be any vector which is capable of replicating autonomously in a given host organism or of integrating into the DNA of the host, typically a plasmid. In preferred embodiments two types of expression vectors for obtaining expression of genes are contemplated. The first contains DNA sequences in which the promoter, gene-coding region, and terminator sequence all originate from the gene to be expressed. Gene truncation may be obtained where desired by deleting undesired DNA sequences (e.g., coding for unwanted domains) to leave the domain to be expressed under control of its own transcriptional and translational regulatory sequences. A selectable marker may also be contained on the vector allowing the selection for integration into the host of multiple copies of the novel gene sequences.

The second type of expression vector is preassembled and contains sequences required for high-level transcription and a selectable marker. It is contemplated that the coding region for a gene or part thereof can be inserted into this general-purpose expression vector such that it is under the transcriptional control of the expression cassettes promoter and terminator sequences.

For example, in Aspergillus, pRAX is such a general-purpose expression vector. Genes or part thereof can be inserted downstream of the strong glaa promoter.

For example, in Hypocrea, pTEX is such a general-purpose expression vector. Genes or part thereof can be inserted downstream of the strong cbh1 promoter.

In the vector, the DNA sequence encoding the CBH2 variant of the present disclosure should be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and signal sequence in reading frame to the structural gene. The promoter may be any DNA sequence that shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. An optional signal peptide provides for extracellular production of the CBH2 variant. The DNA encoding the signal sequence is preferably that which is naturally associated with the gene to be expressed, however the signal sequence from any suitable source, for example an exo-cellobiohydrolase or endoglucanase from Trichoderma, is contemplated in the present disclosure.

The procedures used to ligate the DNA sequences coding for the variant CBH2 of the present disclosure with the promoter, and insertion into suitable vectors are well known in the art.

The DNA vector or construct described above may be introduced in the host cell in accordance with known techniques such as transformation, transfection, microinjection, microporation, biolistic bombardment and the like.

In the preferred transformation technique, it must be taken into account that the permeability of the cell wall to DNA in Hyprocrea sp. (Trichoderma sp.) is very low. Accordingly, uptake of the desired DNA sequence, gene or gene fragment is at best minimal There are a number of methods to increase the permeability of the Hyprocrea sp. (Trichoderma sp.) cell wall in the derivative strain (i.e., lacking a functional gene corresponding to the used selectable marker) prior to the transformation process.

The preferred method in the present disclosure to prepare Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.) for transformation involves the preparation of protoplasts from fungal mycelium. See Campbell et al. Improved transformation efficiency of A. niger using homologous niaD gene for nitrate reductase. Curr. Genet. 16:53-56; 1989. The mycelium can be obtained from germinated vegetative spores. The mycelium is treated with an enzyme that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is preferable to use about a 1.2 M solution of sorbitol in the suspension medium.

Uptake of the DNA into the host strain, (Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.), is dependent upon the calcium ion concentration. Generally between about 10 mM CaCl.sub.2 and 50 mM CaCl.sub.2 is used in an uptake solution. Besides the need for the calcium ion in the uptake solution, other items generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). It is believed that the polyethylene glycol acts to fuse the cell membranes thus permitting the contents of the medium to be delivered into the cytoplasm of the host cell, by way of example either Aspergillus sp. or Hyprocrea sp. strain, and the plasmid DNA is transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Aspergillus sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 10.sup.5 to 10.sup.6/mL, preferably 2.times.10.sup.5/mL are used in transformation. Similarly, a suspension containing the Hyprocrea sp. (Trichoderma sp.) protoplasts or cells that have been subjected to a permeability treatment at a density of 10.sup.8 to 10.sup.9/mL, preferably 2.times.10.sup.8/mL are used in transformation. A volume of 100 .mu.L of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol; 50 mM CaCl.sub.2) are mixed with the desired DNA. Generally a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. However, it is preferable to add about 0.25 volumes to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like may also be added to the uptake solution and aid in transformation.

Generally, the mixture is then incubated at approximately 0.degree. C. for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further enhance the uptake of the desired gene or DNA sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is preferably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then incubated either at room temperature or on ice before the addition of a sorbitol and CaCl.sub.2 solution. The protoplast suspension is then further added to molten aliquots of a growth medium. This growth medium permits the growth of transformants only. Any growth medium can be used in the present disclosure that is suitable to grow the desired transformants. However, if Pyr.sup.+ transformants are being selected it is preferable to use a growth medium that contains no uridine. The subsequent colonies are transferred and purified on a growth medium depleted of uridine.

At this stage, stable transformants may be distinguished from unstable transformants by their faster growth rate and, in Trichoderma, for example, the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium lacking uridine. Additionally, in some cases a further test of stability may made by growing the transformants on solid non-selective medium (i.e. containing uridine), harvesting spores from this culture medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium lacking uridine.

In a particular embodiment of the above method, the CBH2 variant(s) are recovered in active form from the host cell after growth in liquid media as a result of the appropriate post translational processing of the CBH2 variant.

(ii) Yeast

The present disclosure also contemplates the use of yeast as a host cell for CBH2 production. Several other genes encoding hydrolytic enzymes have been expressed in various strains of the yeast S. cerevisiae. These include sequences encoding for two endoglucanases (Penttila et al., Yeast vol. 3, pp 175-185, 1987), two cellobiohydrolases (Penttila et al., Gene, 63: 103-112, 1988) and one beta-glucosidase from Trichoderma reesei (Cummings and Fowler, Curr. Genet. 29:227-233, 1996), a xylanase from Aureobasidlium pullulans (Li and Ljungdahl, Appl. Environ. Microbiol. 62, no. 1, pp. 209-213, 1996), an alpha-amylase from wheat (Rothstein et al., Gene 55:353-356, 1987), etc. In addition, a cellulase gene cassette encoding the Butyrivibrio fibrisolvens endo-[beta]-1,4-glucanase (END1), Phanerochaete chrysosporium cellobiohydrolase (CBH1), the Ruminococcus flavefaciens cellodextrinase (CEL1) and the Endomyces fibrilizer cellobiase (Bgl1) was successfully expressed in a laboratory strain of S. cerevisiae (Van Rensburg et al., Yeast, vol. 14, pp. 67-76, 1998).

C. Introduction of a CBH2-Encoding Nucleic Acid Sequence into Host Cells.

The disclosure further provides cells and cell compositions which have been genetically modified to comprise an exogenously provided variant CBH2-encoding nucleic acid sequence. A parental cell or cell line may be genetically modified (i.e., transduced, transformed or transfected) with a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc, as further described above.

The methods of transformation of the present disclosure may result in the stable integration of all or part of the transformation vector into the genome of the filamentous fungus. However, transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated.

Many standard transfection methods can be used to produce Trichoderma reesei cell lines that express large quantities of the heterologus protein. Some of the published methods for the introduction of DNA constructs into cellulase-producing strains of Trichoderma include Lorito, Hayes, DiPietro and Harman, 1993, Cum Genet. 24: 349-356; Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174; Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6: 155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila and Kolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, for Streptomyces Hopwood et al., 1985, The John Innes Foundation, Norwich, UK and for Bacillus Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, 1990, FEMS Microbiol. Lett. 55: 135-138).

Other methods for introducing a heterologous nucleic acid construct (expression vector) into filamentous fungi (e.g., H. jecorina) include, but are not limited to the use of a particle or gene gun, permeabilization of filamentous fungi cells walls prior to the transformation process (e.g., by use of high concentrations of alkali, e.g., 0.05 M to 0.4 M CaCl.sub.2 or lithium acetate), protoplast fusion or Agrobacterium mediated transformation. An exemplary method for transformation of filamentous fungi by treatment of protoplasts or spheroplasts with polyethylene glycol and CaCl.sub.2 is described in Campbell, E. I. et al., Curr. Genet. 16:53-56, 1989 and Penttila, M. et al., Gene, 63:11-22, 1988.

Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). Also of use is the Agrobacterium-mediated transfection method described in U.S. Pat. No. 6,255,115. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the heterologous gene.

In addition, heterologous nucleic acid constructs comprising a variant CBH2-encoding nucleic acid sequence can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection.

The disclosure further includes novel and useful transformants of filamentous fungi such as H. jecorina and A. niger for use in producing fungal cellulase compositions. The disclosure includes transformants of filamentous fungi especially fungi comprising the variant CBH2 coding sequence, or deletion of the endogenous cbh coding sequence.

Following introduction of a heterologous nucleic acid construct comprising the coding sequence for a variant cbh2, the genetically modified cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying expression of a variant CBH2-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.

The progeny of cells into which such heterologous nucleic acid constructs have been introduced are generally considered to comprise the variant CBH2-encoding nucleic acid sequence found in the heterologous nucleic acid construct.

The disclosure further includes novel and useful transformants of filamentous fungi such as H. jecorina for use in producing fungal cellulase compositions. Aspergillus niger may also be used in producing the variant CBH2. The disclosure includes transformants of filamentous fungi especially fungi comprising the variant cbh 2 coding sequence, or deletion of the endogenous cbh2 coding sequence.

Stable transformants of filamentous fungi can generally be distinguished from unstable transformants by their faster growth rate and, in Trichoderma, for example, the formation of circular colonies with a smooth rather than ragged outline on solid culture medium. Additionally, in some cases, a further test of stability can be made by growing the transformants on solid non-selective medium, harvesting the spores from this culture medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium.

VI. Isolation and Purification of Recombinant CBH2 Protein

In general, a variant CBH2 protein produced in cell culture is secreted into the medium and may be purified or isolated, e.g., by removing unwanted components from the cell culture medium. However, in some cases, a variant CBH2 protein may be produced in a cellular form necessitating recovery from a cell lysate. In such cases the variant CBH2 protein is purified from the cells in which it was produced using techniques routinely employed by those of skill in the art. Examples include, but are not limited to, affinity chromatography (Tilbeurgh et al., FEBS Lett. 16:215, 1984), ion-exchange chromatographic methods (Goyal et al., Bioresource Technol. 36:37-50, 1991; Fliess et al., Eur. J. Appl. Microbiol. Biotechnol. 17:314-318, 1983; Bhikhabhai et al., J. Appl. Biochem. 6:336-345, 1984; Ellouz et al., J. Chromatography 396:307-317, 1987), including ion-exchange using materials with high resolution power (Medve et al., J. Chromatography A 808:153-165, 1998), hydrophobic interaction chromatography (Tomaz and Queiroz, J. Chromatography A 865:123-128, 1999), and two-phase partitioning (Brumbauer, et al., Bioseparation 7:287-295, 1999).

Typically, the variant CBH2 protein is fractionated to segregate proteins having selected properties, such as binding affinity to particular binding agents, e.g., antibodies or receptors; or which have a selected molecular weight range, or range of isoelectric points.

Once expression of a given variant CBH2 protein is achieved, the CBH2 protein thereby produced is purified from the cells or cell culture. Exemplary procedures suitable for such purification include the following: antibody-affinity column chromatography, ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; and gel filtration using, e.g., Sephadex G-75. Various methods of protein purification may be employed and such methods are known in the art and described e.g. in Deutscher, Methods in Enzymology, vol. 182, no. 57, pp. 779, 1990; Scopes, Methods Enzymol. 90: 479-91, 1982. The purification step(s) selected will depend, e.g., on the nature of the production process used and the particular protein produced.

VII. Utility of cbh2 and CBH2

It can be appreciated that the variant cbh nucleic acids, the variant CBH2 protein and compositions comprising variant CBH2 protein activity find utility in a wide variety applications, some of which are described below.

New and improved cellulase compositions that comprise varying amounts BG-type, EG-type and variant CBH-type cellulases find utility in detergent compositions that exhibit enhanced cleaning ability, function as a softening agent and/or improve the feel of cotton fabrics (e.g., “stone washing” or “biopolishing”), in compositions for degrading wood pulp into sugars (e.g., for bio-ethanol production), and/or in feed compositions. The isolation and characterization of cellulase of each type provides the ability to control the aspects of such compositions.

In one approach, the cellulase of the disclosure finds utility in detergent compositions or in the treatment of fabrics to improve the feel and appearance.

Since the rate of hydrolysis of cellulosic products may be increased by using a transformant having at least one additional copy of the cbh gene inserted into the genome, products that contain cellulose or heteroglycans can be degraded at a faster rate and to a greater extent. Products made from cellulose such as paper, cotton, cellulosic diapers and the like can be degraded more efficiently in a landfill. Thus, the fermentation product obtainable from the transformants or the transformants alone may be used in compositions to help degrade by liquefaction a variety of cellulose products that add to the overcrowded landfills.

Separate saccharification and fermentation is a process whereby cellulose present in biomass, e.g., corn stover, is converted to glucose and subsequently yeast strains convert glucose into ethanol. Simultaneous saccharification and fermentation is a process whereby cellulose present in biomass, e.g., corn stover, is converted to glucose and, at the same time and in the same reactor, yeast strains convert glucose into ethanol. Thus, in another approach, the variant CBH type cellulase of the disclosure finds utility in the degradation of biomass to ethanol. Ethanol production from readily available sources of cellulose provides a stable, renewable fuel source.

Cellulose-based feedstocks are comprised of agricultural wastes, grasses and woods and other low-value biomass such as municipal waste (e.g., recycled paper, yard clippings, etc.). Ethanol may be produced from the fermentation of any of these cellulosic feedstocks. However, the cellulose must first be converted to sugars before there can be conversion to ethanol.

A large variety of feedstocks may be used with the inventive variant CBH and the one selected for use may depend on the region where the conversion is being done. For example, in the Midwestern United States agricultural wastes such as wheat straw, corn stover and bagasse may predominate while in California rice straw may predominate. However, it should be understood that any available cellulosic biomass may be used in any region.

The methods of the present disclosure can be used in the production of monosaccharides, disaccharides, and polysaccharides as chemical or fermentation feedstocks for microorganism for the production of organic products, chemicals and fuels, plastics, and other products or intermediates. In particular, the value of processing residues (dried distillers grain, spent grains from brewing, sugarcane bagasse, etc.) can be increased by partial or complete solubilization of cellulose or hemicellulose. In addition to ethanol, some chemicals that can be produced from cellulose and hemicellulose include, acetone, acetate, glycine, lysine, organic acids (e.g., lactic acid), 1,3-propanediol, butanediol, glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, cis, cis-muconic acid, animal feed and xylose.

A cellulase composition containing an enhanced amount of cellobiohydrolase finds utility in ethanol production. Ethanol from this process can be further used as an octane enhancer or directly as a fuel in lieu of gasoline which is advantageous because ethanol as a fuel source is more environmentally friendly than petroleum derived products. It is known that the use of ethanol will improve air quality and possibly reduce local ozone levels and smog. Moreover, utilization of ethanol in lieu of gasoline can be of strategic importance in buffering the impact of sudden shifts in non-renewable energy and petrochemical supplies.

Ethanol can be produced via saccharification and fermentation processes from cellulosic biomass such as trees, herbaceous plants, municipal solid waste and agricultural and forestry residues. However, the ratio of individual cellulase enzymes within a naturally occurring cellulase mixture produced by a microbe may not be the most efficient for rapid conversion of cellulose in biomass to glucose. It is known that endoglucanases act to produce new cellulose chain ends which themselves are substrates for the action of cellobiohydrolases and thereby improve the efficiency of hydrolysis of the entire cellulase system. Therefore, the use of increased or optimized cellobiohydrolase activity may greatly enhance the production of ethanol.

Thus, the inventive cellobiohydrolase finds use in the hydrolysis of cellulose to its sugar components. In one embodiment, a variant cellobiohydrolase is added to the biomass prior to the addition of a fermentative organism. In a second embodiment, a variant cellobiohydrolase is added to the biomass at the same time as a fermentative organism. Optionally, there may be other cellulase components present in either embodiment.

In another embodiment the cellulosic feedstock may be pretreated. Pretreatment may be by elevated temperature and the addition of either of dilute acid, concentrated acid or dilute alkali solution. The pretreatment solution is added for a time sufficient to at least partially hydrolyze the hemicellulose components and then neutralized.

The major product of CBH2 action on cellulose is cellobiose which is available for conversion to glucose by BG activity (for instance in a fungal cellulase product). Either by the pretreatment of the cellulosic biomass or by the enzymatic action on the biomass, other sugars, in addition to glucose and cellobiose, can be made available from the biomass. The hemi-cellulose content of the biomass can be converted (by hemi-cellulases) to sugars such as xylose, galactose, mannose and arabinose. Thus, in a biomass conversion process, enzymatic saccharification can produce sugars that are made available for biological or chemical conversions to other intermediates or end-products. Therefore, the sugars generated from biomass find use in a variety of processes in addition to the generation of ethanol. Examples of such conversions are fermentation of glucose to ethanol (as reviewed by M. E. Himmel et al. pp 2-45, in “Fuels and Chemicals from Biomass”, ACS Symposium Series 666, ed B. C. Saha and J. Woodward, 1997) and other biological conversions of glucose to 2,5-diketo-D-gluconate (U.S. Pat. No. 6,599,722), lactic acid (R. Datta and S-P. Tsai pp 224-236, ibid), succinate (R. R. Gokarn, M. A. Eiteman and J. Sridhar pp 237-263, ibid), 1,3-propanediol (A-P. Zheng, H. Biebl and W-D. Deckwer pp 264-279, ibid), 2,3-butanediol (C. S. Gong, N. Cao and G. T. Tsao pp 280-293, ibid), and the chemical and biological conversions of xylose to xylitol (B. C. Saha and R. J. Bothast pp 307-319, ibid). See also, for example, WO 98/21339.

The detergent compositions of this disclosure may employ besides the cellulase composition (irrespective of the cellobiohydrolase content, i.e., cellobiohydrolase-free, substantially cellobiohydrolase-free, or cellobiohydrolase enhanced), a surfactant, including anionic, non-ionic and ampholytic surfactants, a hydrolase, building agents, bleaching agents, bluing agents and fluorescent dyes, caking inhibitors, solubilizers, cationic surfactants and the like. All of these components are known in the detergent art. The cellulase composition as described above can be added to the detergent composition either in a liquid diluent, in granules, in emulsions, in gels, in pastes, and the like. Such forms are well known to the skilled artisan. When a solid detergent composition is employed, the cellulase composition is preferably formulated as granules. Preferably, the granules can be formulated so as to contain a cellulase protecting agent. For a more thorough discussion, see U.S. Pat. No. 6,162,782 entitled “Detergent compositions containing cellulase compositions deficient in CBH2 type components,” which is incorporated herein by reference.

Preferably the cellulase compositions are employed from about 0.00005 weight percent to about 5 weight percent relative to the total detergent composition. More preferably, the cellulase compositions are employed from about 0.0002 weight percent to about 2 weight percent relative to the total detergent composition.

In addition the variant CBH2 nucleic acid sequence finds utility in the identification and characterization of related nucleic acid sequences. A number of techniques useful for determining (predicting or confirming) the function of related genes or gene products include, but are not limited to, (A) DNA/RNA analysis, such as (1) overexpression, ectopic expression, and expression in other species; (2) gene knock-out (reverse genetics, targeted knock-out, viral induced gene silencing (VIGS, see Baulcombe, 100 Years of Virology, Calisher and Horzinek eds., Springer-Verlag, New York, N.Y. 15:189-201, 1999); (3) analysis of the methylation status of the gene, especially flanking regulatory regions; and (4) in situ hybridization; (B) gene product analysis such as (1) recombinant protein expression; (2) antisera production, (3) immunolocalization; (4) biochemical assays for catalytic or other activity; (5) phosphorylation status; and (6) interaction with other proteins via yeast two-hybrid analysis; (C) pathway analysis, such as placing a gene or gene product within a particular biochemical or signaling pathway based on its overexpression phenotype or by sequence homology with related genes; and (D) other analyses which may also be performed to determine or confirm the participation of the isolated gene and its product in a particular metabolic or signaling pathway, and help determine gene function.

EXPERIMENTAL

The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure

In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); um (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H₂O (water); dH₂O (deionized water); HCl (hydrochloric acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl (sodium chloride); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); CNPG (chloro-nitro-phenyl-beta-D-glucoside); CNP (2-chloro-4-nitrophenol); APB (acid-pretreated bagasse); PASC (phosphoric acid swollen cellulose) PCS (acid-pretreated corn stover); Pi or PI (performance index); HPLC (high pressure liquid chromatography); PAGE (polyacrylamide gel electrophoresis); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); and SEL (site evaluation library).

Example 1 Assays

The following assays were used in the examples described below. Any deviations from the protocols provided below are indicated in the examples. In these experiments, a spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions.

A. Hexokinase Assay for Measurement of Residual Glucose

Residual glucose from H. jecorina culture supernatants expressing CBH2 variants was measured using a hexokinase assay. A volume of 5 μl of supernatant was added to 195 μl glucose hexokinase assay (Instrumentation Laboratory, Breda, Netherlands) in a 96-well microtiterplate (Costar Flat Bottom PS). The plates were incubated at room temperature for 15 min. Following incubation, the absorbance was measured at 340 nm OD. Supernatants of cultures expressing residual glucose were excluded from pooling for further studies.

B. HPLC Assay for Protein Content Determination

The concentration of CBH2 variant proteins from pooled culture supernantants was determined using an Agilent 1100 (Hewlett Packard) HPLC equipped with a Proswift RP 2H column (Dionex). Ten microliters of sample, mixed with 50 μl of 10% acetonitrile in filtered demineralized water was injected following equilibration of the HPLC column with 10% acetonitrile containing 0.01% trifluoroacetic acid. Compounds were eluted using a gradient of 10% to 30% acetonitrile from 0.3 min to 1 min, followed by a gradient of 30% to 65% from 1 min to 4 mins. Protein concentrations of CBH2 variants were determined from a calibration curve generated using purified wild-type CBH2 (6.25, 12.5, 25, 50 μg/ml). To calculate performance index (P_(i) or PI), the ratio of the (average) total protein produced by a variant and (average) total protein produced by the wild-type at the same dose were averaged.

C. Specific Activity Determination by Phosphoric Acid Swollen Cellulose (PASC) Hydrolysis Assay

Cellulose hydrolysis: Phosphoric acid swollen cellulose (PASC) was prepared from Avicel according to a published method (Walseth, Tappi 35:228, 1971; and Wood, Biochem J, 121:353-362, 1971). This material was diluted with buffer and water to achieve a 1% w/v mixture such that the final concentration of sodium acetate was 50 mM, pH 5.0. One hundred microliters of a 1% suspension of PASC in 50 mM sodium acetate buffer (pH5.0) was dispensed in a 96-well microtiterplate (Costar Flat Bottom PS). Ten microliters of a 5 mg/ml culture supernatant from a CBH2 deleted strain was added to the PASC, and 5, 10, 15, or 20 μl of pooled culture supernatants from H. jecorina cells expressing either wild-type CBH2 or CBH2 variants were added to it. Deletion of the CBH2 gene from Hypocrea jecorina (also referred to as Trichoderma reesei) is described in U.S. Pat. Nos. 5,861,271 and 5,650,322. Compensating volumes of acetate buffer were added to make up for differences in total volume. The microtiterplate was sealed and incubated in a thermostatted incubator at 50° C. under continuous shaking at 900 rpm. After two hours, the hydrolysis reaction was stopped by the addition of 100 μl glycine buffer, pH 10 to each well. To calculate performance index (P_(i) or PI), the ratio of the (average) total sugar produced by a variant and (average) total sugar produced by the wild-type at the same dose were averaged. The hydrolysis reaction products were analyzed with the PAHBAH assay. PAHBAH assay: Aliquots of 150 μl of PAHBAH reducing sugar reagent (5% w/v p-hydroxybenzoic acid hydrazide (PAHBAH, Sigma # H9882, dissolved in 0.5 N HCl), (Lever, Anal Biochem, 47:273-279, 1972) were added to all wells of an empty microtiter plate. Ten microliters of the hydrolysis reaction supernatants were added to the PABAH reaction plate. All plates were sealed and incubated at 69° C. under continuous shaking of 900 rpm. After one hour the plates were placed on ice for five minutes and centrifuged at 720×g at room temperature for five minutes. Samples of 80 μL of the developed PAHBAH reaction mixtures were transferred to a fresh (read) plate and absorbance was measured at 410 nm in a spectrophotometer. A cellobiose standard was included as control. A dose response curve was generated for wild-type CBH2 protein.

D. Specific Activity Determination by Hydrolysis of Dilute Acid Pretreated Corn Stover (PCS)

Pretreated Corn Stover (PCS): Corn stover was pretreated with 2% w/w H₂SO₄ as described (Schell et al., J Appl Biochem Biotechnol, 105:69-86, 2003) and followed by multiple washes with deionized water to obtain a paste having a pH of 4.5. Sodium acetate buffer (pH 5.0) was then added to a final concentration of 50 mM sodium acetate and, if necessary, this mixture was then titrated to pH 5.0 using 1N NaOH. The cellulase concentration in the reaction mixture was approximately 7%. Sixty-five microliters of this cellulose suspension was added per well to a 96-well microtiterplate (Nunc Flat Bottom PS). Ten microliters of a 5 mg/ml culture supernatant from a CBH2 deleted strain was added to the PCS, and 5, 10, 15, or 20 μl of pooled culture supernatants from H. jecorina cells expressing either wild-type CBH2 or CBH2 variants were added to it. Compensating volumes of acetate buffer were added to make up for differences in total volume. After sealing of the plate, the plates were placed in a thermostatted incubator at 50° C. under continuous shaking of 1300 rpm for 5 minutes. The plates were then incubated at 50° C. while shaking at 220 rpm under 80% humidity to prevent drying. After 7 days the plates were put on ice for 5 min and the hydrolysis reaction was stopped by the addition of 100 μl glycine buffer, pH 10 to each well. The hydrolysis reaction products were analyzed with the PAHBAH assay. To calculate performance index (P_(i) or PI), the ratio of the (average) total sugar produced by a variant and (average) total sugar produced by the wild-type at the same dose were averaged. PAHBAH assay: Aliquots of 150 μl of PAHBAH reducing sugar reagent (5% w/v p-hydroxybenzoic acid hydrazide (PAHBAH, Sigma # H9882, dissolved in 0.5 N HCl), (Lever, Anal Biochem, 47:273-279, 1972) were added to all wells of an empty microtiter plate. Ten microliters of the hydrolysis reaction supernatants were added to the PABAH reaction plate. All plates were sealed and incubated at 69° C. under continuous shaking of 900 rpm. After one hour the plates were placed on ice for five minutes and centrifuged at 720×g at room temperature for five minutes. Samples of 80 μL of the developed PAHBAH reaction mixtures were transferred to a fresh (read) plate and absorbance was measured at 410 nm in a spectrophotometer. A cellobiose standard was included as control. A dose response curve was generated for wild-type CBH2 protein.

E. Specific Activity Determination by Hydrolysis of Ammonia Pretreated Corncob (CC)

Corn cob was ground to pass through a 0.9 mm screen then pretreated as described in Example 4 of patent application WO2006110901 (herein incorporated by reference for this method). Pretreated corn cob was used as a 7% cellulose suspension in 50 mM sodium acetate pH 5.0. Sixty-five microliters of the suspension was added per well to a 96-well microtiterplate (Nunc Flat Bottom PS). To each well, 20 μl of 3.4 mg/ml supernatant from a strain (Δegl1, Δegl2, Δcbh1, Δcbh2) supplemented with 0.23 mg/ml purified T. reesei beta-glucosidase 1 (Cel3A) was added. Twenty or forty microliters of pooled culture supernatants from H. jecorina cells expressing either wild-type CBH2 or CBH2 variants were added. Compensating volumes of acetate buffer were added to make up for differences in total volume. The plate was incubated at 50° C. while shaking at 220 rpm under 80% humidity to prevent drying. After 3 days the plate was put on ice for 5 min and 100 μl of 100 mM glycine pH 10.0 was added. After mixing, the plate was centrifuged at 3000 rpm for 5 min. A volume of 10 μl supernatant was diluted in 190 μl water. Twenty μl of the diluted solution was transferred to a new 96-well microtiterplate (Costar Flat Bottom PS) containing 100 μl ABTS glucose assay mixture (2.74 mg/ml 2,2′ azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid, 0.333 U/ml horseradish peroxidase type VI, 1 U/ml glucose oxidase) and increase in A₄₂₀ was recorded in a microtiterplate spectrophotometer (Spectramax Plus 384, Molecular Devices). A range of glucose concentrations was included as a standard on each plate (6.25, 12.5, 25, 50, 100, 200, 400 mM). Assays were done in duplicate. A dose response curve was generated for the wild-type CBH2 by fitting the data with the Langmuir equation (y=(x*a)/(x+b)) and the activities of the CBH2 variants were divided by a calculated activity of wild-type CBH2 of the same plate to yield a performance index.

F. Stability of CBH2 Variants in Presence of Ethanol

The stability of wild-type CBH2 and CBH2 variants was tested in the presence of 4.5% ethanol (EtOH) at 49° C. Pooled culture supernatants (80 μL) of H. jecorina cells expressing CBH2 variants were added to a 96-well plate (Greiner V-bottom PS) containing 10 μl of 40.5% EtOH per well. The plates were sealed and incubated in a thermostated incubator at 49° C. for 16 hours with shaking at 900 rpm. Following incubation, the plates were placed on ice for 5 minutes. Residual CBH2 activity was determined using the phosphoric acid swollen cellulose (PASC) hydrolysis assay as described above.

To calculate residual activity, the value of the product formed by the addition of 5, 10, and 20 μl of EtOH-incubated CBH2 to the residual activity PASC assay was divided by the value of the product formed by the addition of 5, 10, 15 and 20 μl of EtOH-free CBH2 to the PASC assay. The individual values of these four ratios were then averaged to give the average residual activity. To determine PI value for the variant, the value of average residual activity for the variants was then divided by the average of the residual activity values of the wild-type CBH2 controls.

G. Thermostability of CBH2 Variants

The thermostability of wild-type CBH2 and CBH2 variants was tested at 53° C. Pooled culture supernatant (80 uL) of H. jecorina cells expressing CBH2 variants were added to a 96-well plate (Greiner V-bottom PS). The plates were sealed and incubated in a thermostatted incubator at 53° C. for 16 hours with shaking at 900 rpm. Following incubation, the plates were placed on ice for 5 minutes. Residual CBH2 activity was determined using the phosphoric acid swollen cellulose (PASC) hydrolysis assay as described above.

To calculate residual activity, the value of the product formed by the addition of 5, 10, 15 and 20 μl of heat-treated CBH2 to the residual activity PASC assay was divided by the value of the product formed by the addition of 5, 10, 15 and 20 μl of unheated CBH2 to the PASC assay. The individual values of these four ratios were then averaged to give the average residual activity. To determine PI value for the variant, the value of average residual activity for the variants was then divided by the average of the residual activity values of the wild-type CBH2 controls.

Example 2 Generation of Hypocrea jecorina CBH2 Site Evaluation and Combinatorial Libraries

The pTTTpyr-cbh2 plasmid containing the Hypocrea jecorina CBH2 protein encoding sequence (SEQ ID NO:1) was sent to BASEClear (Leiden, The Netherlands), GeneArt AG (Regensburg, Germany), and Sloning BioTechnology GmbH (Puchheim, Germany) for the generation of Site Evaluation Libraries (SELs). A map of pTTTpyr-cbh2 is provided in FIG. 3. A request was made to the vendors for the generation of positional libraries at each of the sites in Hypocrea jecorina CBH2 mature protein (SEQ ID NO:3) shown in Table 2-1. The amino acid sequence of CBH2 full length protein is shown in SEQ ID NO:2.

SEQ ID NO:1 sets forth the reference Hypocrea jecorina CBH2 coding DNA sequence:

atgattgtcggcattctcaccacgctggctacgctggccacactcgcagctagtgtgcctctagaggagcggcaagcttgctcaagcgt ctggggccaatgtggtggccagaattggtcgggtccgacttgctgtgatccggaagcacatgcgtctactccaacgactattactccc agtgtcacccggcgctgcaagctcaagctcgtccacgcgcgccgcgtcgacgacttctcgagtatcccccacaacatcccggtcga gctccgcgacgcctccacctggactactactaccagagtacctccagtcggatcgggaaccgctacgtattcaggcaacccattgag gggtcactccagggccaatgcatattacgcctctgaagttagcagcctcgctattcctagcttgactggagccatggccactgctgcag cagctgtcgcaaaggaccctcattatgtggctagatactcttgacaagacccctctcatggagcaaaccaggccgacatccgcaccg ccaacaagaatggcggtaactatgccggacagtagtggtgtatgacttgccggatcgcgattgcgctgccc ttgcctcgaatggcgaa tactctattgccgatggtggcgtcgccaaatataagaactatatcgacaccattcgtcaaattgtcgtggaatattccgatatccggaccct cctggttattgagcctgactctcttgccaacctggtgaccaacctcggtactccaaagtgtgccaatgctcagtcagcctaccttgagtgc atcaactacgccgtcacacagctgaaccaccaaatgagcgatgtataggacgctggccatgcaggatggcaggctggccggcaaa ccaagacccggccgctcagctatttgcaaatgatacaagaatgcatcgtctccgagagctcacgcggattggcaaccaatgtcgcca actacaacgggtggaacattaccagccccccatcgtacacgcaaggcaacgctgtctacaacgagaagctgtacatccacgctattg gacctcacttgccaatcacggctggtccaacgccacttcatcactgatcaaggtcgatcgggaaagcagcctaccggacagcaaca gtggggagactggtgcaatgtgatcggcaccggatttggtattcgcccatccgcaaacactggggactcgttgctggattcgtttgtctg ggtcaagccaggcggcgagtgtgacggcaccagcgacagcagtgcgccacgatttgactcccactgtgcgctcccagatgccttgc aaccggcgcctcaagctggtgcttggttccaagcctactttgtgcagcttctcacaaacgcaaacccatcgttcctgtaa.

SEQ ID NO:2 sets forth the Hypocrea jecorina CBH2 full length protein sequence:

MIVGILTTLATLATLAASVPLEERQACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCL PGAASSSSSTRAASTTSRVSPTTSRSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTPWA NAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANKNGGNY AGQGVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLA NLVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANV YKNASSPRALRGLATNVANYNGWNITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFI TDQGRSGKQPTGQQQWGDWCNVIGTGFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPR FSSHCALPDALQPAPQAGAWFQAYFVQLLTNANPSFL.

SEQ ID NO:3 sets forth the Hypocrea jecorina CBH2 mature protein sequence:

QACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSSSSTRAASTTSRVSPTTS RSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAA AAVAKVPSFMWLDTLDKTPLMEQTLADIRTANKNGGNYAGQFVVYDLPDRDCAALASNGEYS IADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINY AVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSPRALRGLATNVANYNGWN ITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFITDQGRSGKQPTGQQQWGDWCNVIG TGFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPRFDSHCALPDALQPAPQAGAWFQAY FVQLLTNANPSFL.

For each of the 162 sites listed in Table 2-1, an average of 18 substitution variants were generated. The libraries were received as purified plasmids encoding CBH2 variant proteins.

TABLE 2-1 Selected Positions of the Mature CBH2 Protein CBH2 Region Position CBM 5 CBM 18 CBM 19 CBM 28 CBM 30 CBM 32 CBM 35 CBM 38 Linker 63 Linker 77 Linker 79 Linker 80 Catalytic 89 Catalytic 94 catalytic 98 catalytic 100 catalytic 102 catalytic 103 catalytic 104 catalytic 105 catalytic 107 catalytic 111 catalytic 117 catalytic 119 catalytic 120 catalytic 121 catalytic 125 catalytic 126 Catalytic 129 Catalytic 133 Catalytic 134 Catalytic 137 catalytic 138 catalytic 139 catalytic 140 catalytic 141 catalytic 143 catalytic 144 catalytic 147 catalytic 150 catalytic 153 catalytic 154 catalytic 157 catalytic 158 catalytic 161 catalytic 162 catalytic 177 catalytic 178 catalytic 179 catalytic 180 catalytic 181 catalytic 182 catalytic 185 catalytic 186 catalytic 188 catalytic 189 Catalytic 190 Catalytic 191 Catalytic 192 Catalytic 193 Catalytic 194 Catalytic 196 Catalytic 197 Catalytic 201 Catalytic 203 Catalytic 204 Catalytic 206 Catalytic 207 Catalytic 210 Catalytic 214 Catalytic 225 Catalytic 226 Catalytic 228 Catalytic 229 Catalytic 230 Catalytic 231 Catalytic 232 Catalytic 233 Catalytic 234 Catalytic 236 Catalytic 237 Catalytic 239 Catalytic 240 Catalytic 243 catalytic 245 catalytic 247 catalytic 251 catalytic 252 catalytic 254 catalytic 258 catalytic 266 catalytic 267 catalytic 268 catalytic 272 catalytic 274 catalytic 275 catalytic 281 catalytic 285 catalytic 288 catalytic 289 catalytic 291 catalytic 292 catalytic 293 catalytic 294 catalytic 303 catalytic 304 catalytic 306 catalytic 307 catalytic 312 catalytic 313 catalytic 316 catalytic 319 catalytic 322 catalytic 323 catalytic 327 catalytic 328 catalytic 331 catalytic 338 catalytic 339 catalytic 340 catalytic 343 catalytic 344 catalytic 346 catalytic 356 catalytic 360 catalytic 361 catalytic 362 catalytic 363 catalytic 364 catalytic 365 catalytic 371 catalytic 378 catalytic 380 catalytic 381 catalytic 382 catalytic 384 catalytic 386 catalytic 394 catalytic 396 catalytic 399 Catalytic 400 Catalytic 405 Catalytic 406 Catalytic 407 Catalytic 410 Catalytic 413 Catalytic 414 Catalytic 416 Catalytic 417 Catalytic 422 Catalytic 426 Catalytic 427 Catalytic 429 Catalytic 431 Catalytic 433 Catalytic 436 Catalytic 440 Catalytic 441 Catalytic 443 Catalytic 444 Catalytic 445 Catalytic 447

Four synthetic CBH2 combinatorial libraries were also produced by Sloning Biotechnology GmbH (Puchheim, Germany) and BaseClear (Leiden, The Netherlands). Tables 2-2 to 2-5 list the substitutions that could be present in members of the synthetic CBH2 combinatorial libraries (numbered according to the CBH2 mature amino acid sequence).

TABLE 2-2 CBH2 Combinatorial Library 1 Design (RCL 1) Targeted Position Wild-Type Residue Substitution 111 L L, S 144 L L, Q, W 154 T T, C, V 162 Y Y, N 410 R R, S 413 S S, W, Y

TABLE 2-3 CBH2 Combinatorial Library 2 Design (RCL 2) Targeted Position Wild-Type Residue Substitution 98 P P, L 194 K K, C, E 313 S S, T 316 S S, P 384 G G, C, Q 443 N N, I

TABLE 2-4 CBH2 Combinatorial Library 3 Design (RCL 3) Targeted Position Wild-Type Residue Substitution 153 R R, Q 161 N N, A, W 203 R R, H 233 P P, D 422 Q Q, V 444 P P, Q

TABLE 2-5 CBH2 Combinatorial Library 4 Design (RCL 4) Targeted Position Wild-Type Residue Substitution 98 P P, L 111 L L, S 144 L L, W 313 S S, T 316 S S, P 413 S S, W 422 Q Q, V

Production of CBH2 Variant Proteins

Purified pTTTpyr-cbh2 plasmids (p_(cbh1), Amp^(R), Acetamide^(R)) containing open reading frames encoding CBH2 variant sequences were obtained from the vendors specified above. Protoplasts of a quad-deleted H. jecorina strain (Δegl1, Δegl2, Δcbh1 Δcbh2) were transformed with the pTTTpyrG constructs and grown on selective agar containing acetamide at 28° C. for 7 days as described (WO 2009/048488). Genes encoding cellobiohydrolase I (CBHI, Cel7a), cellobiohydrolase II (CBHII, Cel6a), endoglucanase I (EGI, Cel7b), and endoglucanase II (EGII, Cel5a) have been inactivated in the quad-deleted strain. Spores were harvested, replated on acetamide agar, and incubated at 28° C. for 7 days. In addition, spores were harvested in 15% glycerol and stored at −20° C. for further use. For CBH2 variant protein production, a volume of 10 μl spore suspension was added to 200 μl glycine minimal medium supplemented with 2% glucose/sophorose mixture in a PVDF filter plate: 6.0 g/L glycine, 4.7 g/L (NH₄)₂SO₄; 5.0 g/L KH₂PO₄; 1.0 g/L MgSO₄.7H₂O; 33.0 g/L PIPPS; pH 5.5; with post sterile addition of ˜2% glucose/sophorose mixture as the carbon source, 10 ml/L of 100 g/L of CaCl₂, 2.5 ml/L of T. reesei trace elements (400×): 175 g/L Citric acid anhydrous; 200 g/L FeSO₄.7H₂O; 16 g/L ZnSO₄.7H₂O; 3.2 g/L CuSO₄.5H₂O; 1.4 g/L MnSO₄.H₂O; 0.8 g/L H₃BO₃. Each CBH2 variant was grown in quadruplicate. After sealing the plate with an oxygen permeable membrane, the plates were incubated at 28° C. for 6 days, while shaking at 220 rpm. Supernatant was harvested by transferring the culture medium to a microtiter plate under low pressure and tested for residual glucose using the hexokinase assay as described in Example 1.

Example 3 Expression, Activity and Stability of CBH2 Variants

H. jecorina CBH2 SEL and combinatorial variant proteins were tested various properties of interest. In particular, the cellulase variants were tested for protein expression using the HPLC assay (HPLC), specific activity using the PASC hydrolysis assay (Act. PASC) and the PCS hydrolysis assay (Act. PCS), stability in the presence of ethanol (EtOH ratio) and thermostability (heat ratio) as described in Example 1. Combinatorial variants were also tested for specific activity by hydrolysis of ammonia pretreated corncob (Sp. Act. CC) as described in Example 1. Performance data for CBH2 SEL variants are shown in Table 3-1, and performance data for the CBH2 combinatorial variants are shown in Table 3-2. Rows of Table 3-2 lacking performance data correspond to CBH2 combinatorial variants that were not expressed in initial tests.

Performance index (Pi or PI) is the ratio of performance of the variant to the parent or reference cellulase. Various terms set forth below are used to describe the mutation: up mutations have a Pi>1; neutral mutations have a Pi>0.5, non-deleterious mutations have a Pi>0.05; deleterious mutations have a Pi=0.05; combinable mutations are those mutations for which the variant has PI values=0.5 for at least one property, and >0.05 for all properties. Combinable mutations are mutations that can be combined to deliver proteins with appropriate PIs for one or more desired properties. Positions at which mutations occur are classed as follows: Non-restrictive positions have ≧20% neutral mutations for at least one property; and Restrictive positions have <20% neutral mutations for activity and stability.

This data may be used to engineer any CBH. Even if the CBH to be engineered has an amino acid different from that of CBH2 at a particular position, this data may be used to find a substitution that will alter the desired properties by identifying the best choice substitution, including substitution to the CBH2 wild type amino acid

Table 3-1 shows performance index values (Pi or PI) for 2,828 variants of Hypocrea jecorina CBH2 at 162 positions. Performance indices less than or equal to 0.05 were fixed to 0.05 and indicated in bold italics in the table.

TABLE 3-1 Performance Index Values for CBH2 Variants PASC PCS Res. Res. Position Variant HPLC Sp. Ac. Sp. Ac. EtOH Heat 5 S005A 1.73 1.06 1.01 0.99 0.95 5 S005C 0.34 1.03 0.92 0.95 0.76 5 S005D 0.08 0.69 0.44 1.87 2.70 5 S005E 0.82 1.10 1.00 0.85 0.63 5 S005F 0.35 2.69 0.87 0.99 0.98 5 S005G 0.73 1.06 0.84 0.97 0.89 5 S005H 0.14 0.77 0.50 1.06 0.68 5 S005I 1.35 0.57 0.54 1.77 1.07 5 S005K 0.52 0.98 0.68 0.97 0.94 5 S005L 1.25 1.10 0.99 0.99 0.96 5 S005M 0.44 1.11 0.82 0.96 0.89 5 S005N 0.28 0.74 0.41 1.78 3.67 5 S005P 0.46 0.66 0.63 1.46 1.25 5 S005Q 0.72 1.17 0.98 0.94 0.92 5 S005R 0.36 1.01 0.73 0.89 0.82 5 S005T 0.28 1.05 0.73 0.98 0.84 5 S005V 0.40 1.15 0.79 0.91 0.85 5 S005W 0.61 1.11 1.00 0.95 0.87 5 S005Y 0.98 1.08 0.84 0.95 0.96 18 P18A 0.57 1.11 1.05 0.94 0.90 18 P18C 0.16 0.63 0.48 1.14 1.09 18 P18D 0.20 0.66 0.93 0.52 1.20 18 P18E 0.51 0.87 0.42 1.01 1.09 18 P18F 0.22 0.69 0.37 1.02 0.92 18 P18G 0.88 1.03 0.57 0.31 0.18 18 P18H 0.88 1.08 0.89 0.92 0.98 18 P18I 0.77 1.00 0.89 0.88 0.90 18 P18K 0.24 0.50 0.39 2.14 2.11 18 P18L 0.56 1.14 0.77 0.91 0.91 18 P18M 1.38 0.98 0.84 0.97 1.01 18 P18N 0.24 0.46 0.35 4.62 7.95 18 P18Q 0.12 0.28

10.15  19.36  18 P18R 0.39 0.98 0.75 0.93 0.81 18 P18S 0.88 1.12 0.86 0.94 1.02 18 P18T 0.46 0.69 0.45 0.85 1.14 18 P18V 0.62 0.95 0.93 0.93 0.89 18 P18W 0.70 1.09 0.98 0.92 0.93 18 P18Y 0.91 1.08 0.91 0.91 0.95 19 T19A 0.96 1.06 0.96 0.89 0.87 19 T19C 0.08 0.12

3.22 3.56 19 T19E 0.68 1.17 0.87 0.88 0.89 19 T19F 0.75 1.15 0.91 0.92 0.95 19 T19G 1.92 1.03 0.99 0.94 1.00 19 T19I 1.08 1.19 1.01 0.85 0.95 19 T19K 1.01 1.25 1.00 0.91 0.94 19 T19L 0.45 1.18 0.92 0.83 0.85 19 T19M 1.71 1.04 0.80 0.89 1.04 19 T19N 0.11 0.20 0.07 4.18 10.48  19 T19P 0.77 1.08 1.08 0.87 0.98 19 T19Q 0.67 1.07 0.84 0.92 0.95 19 T19R 1.11 1.08 0.89 0.91 1.04 19 T19S 0.81 1.13 0.84 0.87 0.95 19 T19V 0.09 0.18 0.09 13.65  56.16  19 T19W 0.25 0.51 0.47 0.77 0.59 19 T19Y 0.69 1.08 0.97 0.85 0.92 28 V28A 0.55 0.97 1.10 0.99 0.94 28 V28C 0.82 1.00 0.96 0.92 0.71 28 V28D 0.11

0.29 1.43 6.68 28 V28E 0.43 0.90 0.75 0.96 0.94 28 V28F 0.47 0.93 0.71 0.95 0.95 28 V28G 0.88 0.92 0.84 0.95 0.99 28 V28H 0.08

0.15 5.35 18.58  28 V28I 1.01 1.02 0.75 1.00 1.00 28 V28K 0.67 1.03 0.88 0.96 0.96 28 V28L 0.86 1.02 0.83 0.92 1.05 28 V28M 0.71 1.02 0.71 0.95 0.98 28 V28N 0.72 0.53 0.65 0.81 0.97 28 V28P 0.80 0.52 0.72 0.83 0.93 28 V28Q 0.56 0.97 0.73 0.95 0.98 28 V28R 0.76 1.01 0.74 0.95 0.98 28 V28S 0.52 0.96 0.88 0.97 0.98 28 V28T 0.52 1.03 0.84 1.02 1.00 28 V28W 0.42 0.84 0.75 0.95 0.90 28 V28Y 0.51 0.95 0.83 0.95 0.90 30 S30A 1.09 1.06 1.01 1.08 1.06 30 S30C 1.26 0.99 0.96 1.08 1.09 30 S30D 0.30 0.57 0.50 0.57 0.94 30 S30E 0.14 0.44 0.25 0.69 1.00 30 S30F 0.58 0.91 0.67 0.96 0.97 30 S30G 0.70 0.98 0.72 1.10 1.02 30 S30H 0.30 0.61 0.56 0.84 2.68 30 S30I 0.66 1.02 0.72 1.03 1.02 30 S30K 1.31 0.94 0.70 1.12 1.16 30 S30L 0.46 0.93 0.44 0.93 0.85 30 S30M 0.81 1.03 0.89 1.14 1.00 30 S30N 0.19 0.57 0.42 0.95 1.12 30 S30P 0.31 0.42 0.50 0.88 1.02 30 S30Q 0.63 0.65 0.43 0.85 1.65 30 S30R 0.50 0.67 0.45 1.11 1.37 30 S30T 0.81 0.99 0.76 1.09 1.05 30 S30V 0.76 1.04 0.73 1.02 1.02 30 S30W 0.41 0.92 0.59 0.93 0.78 30 S30Y 0.21 0.52 0.40 1.11 1.14 32 D32A 0.51 1.10 1.32 1.00 1.10 32 D32C 0.24 0.93 1.02 0.92 0.89 32 D32E 0.44 1.31 0.42 0.44 0.47 32 D32F 0.18 0.65 0.45 0.77 0.93 32 D32G 0.32 1.17 1.08 0.97 0.92 32 D32H 0.41 0.65 0.63 0.78 1.12 32 D32I 0.64 1.10 1.06 0.99 1.00 32 D32K 0.39 1.00 0.77 0.95 0.93 32 D32L 0.43 1.10 1.17 0.94 1.02 32 D32M 0.37 1.14 0.96 0.90 0.91 32 D32N 0.61 0.73 0.55 0.94 1.12 32 D32P 0.49 0.99 0.80 0.59 0.56 32 D32Q 0.65 1.11 1.00 0.94 0.97 32 D32R 0.28 1.05 0.78 0.87 0.87 32 D32S 0.31 0.91 0.78 0.91 0.87 32 D32T 0.38 0.74 0.67 1.00 1.10 32 D32V 0.49 1.01 0.96 1.02 1.04 32 D32W 0.69 0.91 0.93 0.97 0.99 32 D32Y 0.82 1.02 1.04 1.00 1.03 35 S35A 0.86 0.92 0.87 1.05 0.92 35 S35C 0.59 0.84 0.99 0.92 0.68 35 S35E 0.29 0.91 0.98 1.06 0.94 35 S35F 0.08 1.17 0.90 1.02 0.89 35 S35G 0.67 0.88 0.76 1.03 0.93 35 S35I 0.58 0.61 0.65 0.78 0.70 35 S35L 0.37 0.93 1.02 0.92 0.85 35 S35M 0.57 0.95 1.01 0.96 0.87 35 S35N 0.42 0.88 1.04 1.00 1.01 35 S35P 0.55 0.48 0.74 0.82 0.86 35 S35Q 0.53 0.90 0.88 0.98 0.94 35 S35R 0.38 0.91 0.99 0.87 0.83 35 S35V 0.89 0.65 0.57 0.86 0.93 35 S35W 1.60 1.01 1.03 1.10 1.05 35 S35Y 0.64 0.97 0.85 0.87 0.79 38 L38A 0.64 0.86 0.96 1.06 1.00 38 L38E 0.65 0.94 0.98 0.95 1.02 38 L38P 0.22 0.23 0.42 0.91 0.72 38 L38S 0.53 0.84 0.71 0.99 0.91 38 L38W 0.30 0.51 0.77 0.84 0.53 63 R63A 1.21 1.03 0.94 0.97 1.00 63 R63C 1.47 1.02 1.05 0.96 1.06 63 R63D 0.12 0.11 0.19

1.02 63 R63E 0.88 0.94 0.98 0.96 1.05 63 R63F 0.91 0.95 0.85 0.94 1.09 63 R63G 0.71 0.96 0.95 1.08 1.20 63 R63H 0.68 0.94 0.92 1.04 1.00 63 R63I 0.81 0.93 1.11 1.03 1.09 63 R63K 0.23 0.53 0.77 0.81 0.91 63 R63L 0.94 0.96 0.91 1.06 1.29 63 R63M 1.22 0.99 1.10 1.05 1.09 63 R63N 1.23 1.02 1.11 1.03 1.07 63 R63P 0.96 0.99 1.17 1.04 1.09 63 R63Q 0.87 0.99 1.01 0.99 1.13 63 R63S 0.59 0.92 1.09 1.00 0.94 63 R63T 0.43 0.87 0.94 0.91 0.84 63 R63V 1.00 0.99 0.89 1.03 1.06 63 R63W 0.98 0.97 0.91 1.08 1.10 63 R63Y 1.29 1.03 0.99 1.06 1.07 77 R77F 0.25 0.81 0.58 0.92 0.81 77 R77G 0.41 1.02 0.75 1.00 0.93 77 R77L 0.14 0.53 0.36 0.94 0.79 77 R77N 0.83 0.99 0.87 1.00 1.02 79 P79C 0.96 1.09 0.93 1.15 0.92 79 P79D 0.33 1.08 1.17 1.02 0.94 79 P79E 0.64 1.11 0.96 1.05 0.98 79 P79F 0.27 0.79 0.74 1.00 0.91 79 P79H 0.21 0.76 0.68 0.92 0.77 79 P79I 0.37 0.91 0.98 0.93 0.93 79 P79K 0.16 1.09 0.62 0.79 0.70 79 P79L 0.66 1.12 0.97 1.10 1.09 79 P79M 0.74 0.94 1.04 1.06 1.06 79 P79N 0.74 1.10 0.94 1.04 0.96 79 P79R 0.41 1.11 0.86 1.04 0.97 79 P79S 0.89 1.04 0.86 1.20 1.02 79 P79V 0.32 0.87 0.84 0.97 0.99 79 P79W 0.53 1.02 1.09 1.05 0.98 80 P80C 0.85 1.11 1.04 1.14 0.92 80 P80D 1.19 1.07 1.14 1.26 1.08 80 P80E 0.41 0.88 0.86 1.14 0.88 80 P80F 0.25 1.07 0.85 0.97 0.92 80 P80G 0.87 1.10 1.05 1.14 0.97 80 P80H 0.33 0.83 0.83 1.11 0.90 80 P80I 0.30 0.84 0.74 0.90 0.88 80 P80K 0.52 0.71 0.53 1.06 1.01 80 P80L 1.23 1.09 0.96 1.09 1.05 80 P80Q 0.77 1.10 0.87 1.06 1.05 80 P80R 0.25 1.04 1.23 1.06 0.96 80 P80T 0.90 0.88 0.99 1.94 1.80 80 P80V 0.36 0.99 1.06 0.93 0.92 80 P80W 1.13 1.01 0.95 1.21 1.08 89 S89A 0.19 0.70 1.04 0.73 0.50 89 S89C 0.27 0.82 1.09 0.83 0.90 89 S89D 1.45 1.06 1.13 1.05 1.02 89 S89E 0.28 0.87 0.87 0.94 0.74 89 S89F 0.26 0.79 0.79 0.97 0.84 89 S89G 0.27 0.82 0.82 0.88 0.78 89 S89H 1.57 1.03 0.86 1.02 1.04 89 S89I 0.51 1.01 0.92 1.03 1.07 89 S89K 0.51 0.93 0.76 0.99 0.96 89 S89L 0.69 1.02 0.97 0.90 0.87 89 S89M 0.22 0.77 1.02 0.85 0.92 89 S89N 0.28 0.85 0.68 0.83 0.88 89 S89P 0.64 0.98 1.18 0.99 0.92 89 S89Q 0.51 0.80 0.53 0.98 1.15 89 S89R 0.57 1.00 0.83 1.03 0.94 89 S89T 0.88 1.03 0.95 1.02 1.03 89 S89V 0.16 0.65 1.02 0.73 0.61 89 S89W 0.82 1.03 1.02 0.92 0.79 89 S89Y 0.70 1.02 0.97 1.00 1.00 94 V94A 1.18 0.98 1.45 1.11 1.06 94 V94C 0.38 0.79 0.66 0.97 0.76 94 V94D 1.13 0.99 0.92 1.10 0.97 94 V94F 0.28 0.73 0.55 0.89 0.89 94 V94G 0.25 0.68 0.43 0.93 0.79 94 V94H 0.23 0.64 0.52 1.03 0.97 94 V94K 0.50 0.98 0.73 1.07 1.08 94 V94P 0.13 0.14 0.17 0.16 1.34 94 V94R 0.22 0.90 0.58 0.76 1.03 94 V94S 0.24 0.76 0.44 0.85 0.77 94 V94T 0.22 0.80 0.37 0.71 0.86 98 P98A 0.90 1.09 0.94 1.01 0.99 98 P98C 0.53 1.01 1.23 1.06 1.03 98 P98D 0.28 0.26 0.35 1.32 2.58 98 P98E 0.30 0.54 0.35 0.99 1.28 98 P98F 0.30 0.77 0.79 0.67 0.75 98 P98G 0.61 0.97 0.71 0.92 1.00 98 P98H 0.29 0.67 0.35 0.62 0.87 98 P98I 0.51 1.05 0.84 1.15 0.88 98 P98K 0.29 0.30 0.23 1.06 2.07 98 P98L 0.80 1.12 0.89 1.16 1.45 98 P98M 0.58 1.03 0.91 1.05 0.92 98 P98N 0.33 0.42 0.38 1.00 1.12 98 P98Q 0.50 1.03 0.90 1.14 1.46 98 P98R 0.28 0.30 0.05 0.93 1.55 98 P98S 0.32 0.27 0.16 1.23 1.36 98 P98T 0.19 0.32

1.31 1.98 98 P98V 0.60 1.06 1.08 1.05 1.08 98 P98W 0.37 0.38 0.34 0.82 1.01 98 P98Y 0.25 0.65 0.19 0.87 0.93 100 A100C 0.37 0.91 0.83 0.71 0.61 100 A100D 0.14 0.38 0.27 0.09 0.39 100 A100E 0.18 0.67 0.48 0.51 0.22 100 A100F 0.11 0.34 0.27 0.42 0.74 100 A100G 0.26 0.79 0.54 0.36 0.18 100 A100H 0.11 0.09 0.14 1.53 0.51 100 A100K 0.16 0.97 0.59 0.71 0.65 100 A100L 0.25 0.78 0.49 0.62 0.42 100 A100N 0.31 0.92 0.70 0.53 0.26 100 A100P 0.14 0.65 0.53 0.63 0.44 100 A100R 0.14 0.37 0.26 0.53 0.81 100 A100S 0.33 0.90 0.64 0.68 0.48 100 A100T 0.39 0.87 0.70 0.86 0.89 100 A100V 0.13 0.77 0.48 0.93 0.61 100 A100W 0.59 0.92 0.60 0.62 0.29 100 A100Y 0.25 0.65 0.62 0.81 0.42 102 A102C 0.11 0.05 0.20 1.02 5.64 102 A102D 0.60 0.98 1.01 0.82 0.64 102 A102E 0.77 0.98 0.69 1.01 1.01 102 A102F 0.57 0.97 0.86 0.91 0.81 102 A102G 0.55 1.00 0.90 0.93 0.89 102 A102H 0.96 1.05 0.87 0.91 0.96 102 A102I 0.43 0.88 0.73 0.77 0.75 102 A102K 0.50 0.97 0.91 0.95 1.02 102 A102L 0.58 0.99 0.82 0.91 0.86 102 A102M 0.56 0.94 0.75 0.93 0.87 102 A102N 0.62 1.01 0.75 0.93 0.92 102 A102P 0.45 0.87 0.63 0.80 0.50 102 A102Q 0.10 0.09 0.14 1.47 2.14 102 A102R 0.55 1.02 0.83 0.99 1.02 102 A102S 0.51 0.99 0.84 0.95 0.96 102 A102T 0.49 0.96 0.81 0.69 0.54 102 A102V 0.49 0.95 0.74 0.73 0.55 102 A102W 0.11 0.06

44.39  31.37  102 A102Y 0.55 0.97 0.87 0.84 0.72 103 Y103A 0.89 1.01 1.02 0.64 0.45 103 Y103C 0.38 0.76 0.55 0.63 0.64 103 Y103D 0.62 0.97 0.40 0.21 0.10 103 Y103E 0.90 0.95 0.92 0.92 0.61 103 Y103F 1.32 0.93 0.88 1.01 0.98 103 Y103G 0.54 0.85 0.50 0.47 0.12 103 Y103H 1.18 0.96 0.98 1.12 1.15 103 Y103I 0.09 0.12

0.31 1.44 103 Y103K 0.83 0.90 0.54 0.96 0.99 103 Y103L 1.03 1.02 0.94 0.88 0.66 103 Y103M 0.08 0.09 0.09 0.53 0.91 103 Y103P 0.51 0.85 0.47 0.44 0.21 103 Y103Q 0.81 0.96 0.48 0.90 0.66 103 Y103R 0.82 0.88 0.67 1.00 0.99 103 Y103S 0.42 0.85 0.56 0.50 0.17 103 Y103T 0.41 0.74 0.33 0.44 0.42 103 Y103V 1.02 0.93 0.63 0.95 0.87 103 Y103W 0.93 0.96 0.72 0.75 0.56 104 Y104A 0.36 0.82 0.27 0.32 0.17 104 Y104C 0.19 0.51

0.31 0.18 104 Y104D 0.23 0.44 0.14 0.40 0.22 104 Y104E 0.12 0.05

4.53 29.82  104 Y104F 0.92 1.02 0.66 0.49 0.12 104 Y104G 0.28 0.50

0.35 0.28 104 Y104H 0.12 0.10

2.55 1.45 104 Y104I 0.27 0.61 0.14 0.35 0.16 104 Y104K 0.38 0.45 0.15 0.24 0.21 104 Y104L 0.50 0.87 0.23 0.23 0.13 104 Y104M 0.41 0.73 0.26 0.27 0.15 104 Y104N 0.22 0.15 0.14 1.06 1.75 104 Y104P 0.22 0.52 0.11 0.60 0.25 104 Y104Q 0.20 0.08

1.63 1.16 104 Y104R 0.37 0.80 0.45 0.15 0.14 104 Y104S 0.38 0.48 0.26 0.46 0.17 104 Y104T 0.21 0.51 0.18 0.31 0.21 104 Y104V 0.23 0.28 0.09 0.15 0.47 104 Y104W 0.77 0.91 0.60 0.37 0.16 105 A105D 0.17 0.36 0.58 0.20 0.05 105 A105G 0.21 0.55 0.64 0.89 0.52 105 A105P 0.11 0.16 0.30 0.14 0.71 105 A105R 0.42 0.97 0.89 1.02 1.01 105 A105T 0.34 0.92 0.72 0.51 0.18 105 A105V 0.35 0.78 0.74 0.60 0.24 105 A105Y 0.44 0.56 0.41 0.09 0.13 107 E107D 0.19 0.27 0.40 0.07 0.23 107 E107I 0.78 0.82 0.40 0.15 0.10 107 E107K 0.23 0.46 0.53 0.57 0.31 107 E107N 0.38 0.62 0.60 0.09 0.16 107 E107P 0.26 0.42 0.25

0.26 107 E107Q 1.19 0.94 0.54 0.58 0.14 107 E107R 0.25 0.53 0.21

0.08 107 E107S 0.89 0.90 0.39 0.06 0.12 107 E107T 0.53 0.72 0.55

0.09 107 E107V 1.46 1.00 0.47 0.06 0.08 107 E107W 0.69 0.73 0.37 0.06 0.18 107 E107Y 0.88 0.82 0.37

0.08 111 L111A 1.36 1.06 1.25 0.84 0.30 111 L111C 0.57 0.81 0.99 1.00 0.93 111 L111D 0.28 0.65 0.54 0.67 0.48 111 L111E 0.69 1.35 1.34 0.91 0.75 111 L111F 0.52 0.67 0.49 1.02 0.76 111 L111G 0.52 1.18 0.88 0.58 0.19 111 L111H 0.55 0.90 0.80 0.86 0.82 111 L111I 0.93 0.77 0.90 0.86 0.80 111 L111K 0.43 0.90 0.68 0.94 0.68 111 L111M 1.99 1.19 0.97 1.00 0.82 111 L111N 0.26 0.76 0.80 0.73 0.53 111 L111P 0.19 0.49 0.29

111 L111Q 0.96 1.34 1.11 0.95 0.82 111 L111R 0.52 1.11 0.84 0.84 0.56 111 L111S 0.61 0.92 0.92 1.00 1.04 111 L111T 0.66 1.17 0.84 0.81 0.55 111 L111V 0.73 0.76 0.62 0.53 0.23 111 L111W 0.52 0.89 0.77 0.68 0.41 111 L111Y 0.45 0.75 0.57 1.11 0.84 117 T117A 0.96 1.07 0.80 0.85 0.85 117 T117C 0.61 0.94 0.94 0.87 0.96 117 T117D 0.95 0.99 1.08 0.94 0.85 117 T117E 0.62 1.14 0.97 1.01 1.03 117 T117F 0.51 1.14 0.82 0.88 0.90 117 T117G 0.63 1.01 0.81 0.73 0.64 117 T117H 0.12 0.23 0.51 3.12 8.65 117 T117K 0.69 1.03 0.82 0.95 1.05 117 T117L 0.51 0.99 1.00 0.80 0.77 117 T117M 0.62 0.99 1.02 0.92 0.88 117 T117N 1.35 1.03 0.84 0.99 0.98 117 T117P 0.32 0.88 0.62 0.69 0.46 117 T117Q 0.28 0.93 0.71 0.95 0.99 117 T117R 0.60 1.04 0.78 0.94 0.93 117 T117S 0.95 1.03 0.93 1.06 1.16 117 T117V 1.00 1.03 0.70 0.92 0.94 117 T117W 0.72 1.01 0.66 0.80 0.74 117 T117Y 1.18 1.05 0.84 0.95 0.91 119 A119C 0.28 1.02 0.98 0.86 0.61 119 A119D 0.31 0.84 0.79 0.67 0.57 119 A119E 0.25 1.16 0.99 0.86 0.84 119 A119F 0.28 0.89 0.60 0.68 0.58 119 A119G 0.69 0.97 0.86 0.89 0.73 119 A119H 0.49 1.03 0.85 0.91 0.71 119 A119I 0.31 0.74 0.62 0.77 0.62 119 A119K 0.33 0.89 0.65 1.00 0.72 119 A119L 0.55 1.11 0.94 0.84 0.59 119 A119M 0.30 0.71 0.36 0.69 0.77 119 A119N 0.85 1.04 0.66 1.00 0.84 119 A119P 0.71 1.02 1.10 1.07 0.97 119 A119Q 0.58 1.17 0.82 1.09 0.88 119 A119R 0.56 0.80 0.42 0.92 0.88 119 A119S 1.44 0.99 0.97 1.05 0.53 119 A119T 0.80 0.86 0.76 0.94 0.68 119 A119V 0.36 0.89 0.45 0.90 0.91 119 A119W 0.38 0.95 0.78 0.61 0.52 119 A119Y 0.38 0.54 0.47 0.79 0.99 120 M120A 0.51 1.00 0.73 0.51 0.28 120 M120C 0.52 0.88 0.74 0.66 0.58 120 M120D 0.31 0.95 0.76 0.32 0.31 120 M120E 0.18 0.43 0.09 1.51 1.97 120 M120F 0.53 1.04 0.70 0.87 0.80 120 M120G 0.35 0.49 0.30 0.92 1.21 120 M120H 0.34 0.70 0.54 0.77 1.16 120 M120I 0.27 0.85 0.44 0.74 0.70 120 M120K 0.34 1.10 0.49 0.72 0.76 120 M120L 1.64 1.08 0.68 1.07 1.17 120 M120N 0.17 0.35

0.40 0.62 120 M120P 0.28 0.53 0.33 0.65 0.71 120 M120Q 0.30 0.61 0.34 1.36 1.41 120 M120R 0.31 0.80 0.14 0.31 0.28 120 M120S 0.40 1.04 0.58 0.64 0.31 120 M120T 0.35 0.95 0.52 0.67 0.45 120 M120V 0.30 0.60 0.47 0.91 1.12 120 M120W 0.53 1.06 0.77 1.01 0.98 120 M120Y 0.70 0.84 0.47 0.91 0.81 121 A121C 0.17 0.79 0.59 0.64 0.38 121 A121D 0.08 1.27 0.35 0.31 0.22 121 A121E 0.11 0.30

0.99 1.43 121 A121F 0.23 0.68 0.56 0.30 0.24 121 A121G 0.13 0.49 0.37 14.85  14.78  121 A121H 0.08 0.39 0.49 0.36 0.28 121 A121I 0.12 0.39 0.32 1.82 1.58 121 A121K 0.23 0.78 0.62 0.95 0.91 121 A121L 0.40 0.66 0.46 0.76 0.76 121 A121M 0.11 0.85 0.48 0.85 0.53 121 A121N 0.09 0.54 0.15 0.56 0.59 121 A121P 0.10 0.30 0.18 1.03 1.05 121 A121Q

121 A121R 0.52 1.10 0.85 0.98 1.01 121 A121S 0.14 0.74 0.85 0.60 0.24 121 A121T 0.25 0.62 0.46 0.49 0.27 121 A121V 0.15 0.73 0.57 0.81 0.75 121 A121W 0.33 0.46 0.45 0.26 0.31 121 A121Y 0.35 0.77 0.57 0.36 0.16 125 A125C 0.50 0.37 0.23 0.39 0.71 125 A125D 0.13 0.46 0.21 1.24 2.42 125 A125E 0.70 0.66 0.40 0.96 1.07 125 A125F 0.42 0.86 0.50 0.29 0.39 125 A125G 0.17 0.50 0.10 1.45 2.19 125 A125H 0.46 1.06 0.44 0.39 0.30 125 A125I 0.31 0.60 0.37 0.91 1.27 125 A125K 0.60 1.12 0.76 0.71 0.62 125 A125L 0.36 1.09 0.38 0.69 0.60 125 A125M 0.60 1.01 0.64 0.79 0.71 125 A125N 0.40 0.97 0.49 0.46 0.34 125 A125P 0.43 0.55 0.33 0.31 0.45 125 A125Q 0.29 0.74 0.34 0.78 0.73 125 A125R 0.56 1.09 0.82 0.74 0.69 125 A125S 0.65 0.93 0.47 0.87 0.71 125 A125T 0.50 1.00 0.43 0.50 0.28 125 A125V 0.46 0.94 0.60 0.54 0.50 125 A125W 0.50 0.94 0.49 0.28 0.13 125 A125Y 0.34 0.63 0.43 0.61 0.91 126 A126D 0.22 0.80 0.45 0.23 0.31 126 A126E 1.46 1.02 0.84 1.06 1.04 126 A126F 0.61 0.88 0.89 0.89 0.66 126 A126G 0.19 0.46 0.67 0.80 0.67 126 A126H 0.27 0.86 1.16 0.84 0.75 126 A126I 0.23 0.65 0.41 0.37 0.26 126 A126K 0.80 1.00 0.86 1.10 1.15 126 A126L 0.44 1.10 0.89 0.88 0.63 126 A126M 0.30 0.67 0.71 0.95 0.83 126 A126N 0.79 0.92 0.78 1.04 0.75 126 A126P 0.17 0.26 0.62 1.39 0.83 126 A126Q 1.31 0.96 0.97 1.11 1.05 126 A126R 0.82 0.88 0.82 0.97 0.95 126 A126S 0.58 0.95 0.80 1.11 1.00 126 A126T 0.59 0.49 0.60 0.65 0.56 126 A126V 0.62 0.96 0.68 0.97 0.70 126 A126W 0.50 0.85 0.95 0.65 0.52 126 A126Y 0.75 0.96 0.80 0.82 0.68 129 K129A 0.23 1.03 0.81 0.74 0.57 129 K129L 0.80 0.94 0.80 0.82 0.71 129 K129N 0.15 0.93 0.65 0.73 0.61 129 K129Q 0.41 1.30 0.89 1.01 0.99 129 K129S 0.37 1.05 0.92 0.84 0.84 129 K129T 0.16 0.87 0.37 0.81 0.69 129 K129V 0.17 0.93 0.91 0.63 0.45 129 K129Y 0.35 0.95 0.82 0.68 0.46 133 F133A 0.19 0.25 0.13 0.84 0.80 133 F133C 0.15 0.20 0.09 0.71 1.32 133 F133D 0.16 0.16 0.20 0.26 2.20 133 F133G 0.17 0.12 0.19 0.87 5.31 133 F133H 0.14 0.22 0.06 0.90 0.76 133 F133K 0.11 0.16

0.64 8.77 133 F133P 0.12 0.24

0.33 0.41 133 F133Q 0.12 0.26 0.16 0.58 1.12 133 F133S 0.12 0.27 0.05 1.08 2.34 133 F133T 0.11 0.24

0.21 0.52 133 F133W 0.13 0.52 0.27 0.42 0.21 133 F133Y 0.13 0.60 0.25 0.53 0.74 134 M134A 1.09 0.96 1.06 0.79 0.76 134 M134C 0.91 1.01 0.84 0.80 1.20 134 M134D 0.42 0.69 0.50 0.89 1.52 134 M134E 0.82 1.10 0.94 0.88 0.97 134 M134F 1.53 1.12 0.91 1.02 1.19 134 M134G 0.33 1.11 0.80 1.16 1.47 134 M134H 0.93 1.17 0.66 0.79 0.53 134 M134I 1.16 1.13 0.72 1.20 1.10 134 M134K 0.53 0.56 0.38 2.39 3.02 134 M134L 1.45 1.15 0.75 1.67 1.64 134 M134N 0.90 1.25 0.93 1.09 0.76 134 M134P 0.37 0.40 0.28 2.37 3.64 134 M134Q 0.57 1.27 0.83 0.99 0.93 134 M134R 0.84 1.54 0.86 1.74 1.18 134 M134S 0.55 1.15 0.70 1.31 1.40 134 M134T 0.65 1.54 0.64 1.33 1.29 134 M134V 1.50 1.24 0.69 1.74 1.84 134 M134W 0.39 0.93 0.57 0.98 1.47 134 M134Y 0.93 1.15 0.68 1.48 0.96 137 D137A 0.56 0.95 0.80 1.06 1.06 137 D137C 0.35 0.31 0.30 0.92 1.04 137 D137E 0.48 0.85 0.50 1.00 0.93 137 D137F 0.28 0.46 0.44 0.80 0.88 137 D137G 0.23 0.49 0.39 1.24 1.25 137 D137H 0.14 0.24

1.77 1.76 137 D137I 0.20 0.28 0.19 0.96 0.85 137 D137K 0.29 0.30 0.17 0.72 1.06 137 D137L 0.23 0.21 0.31 0.90 1.53 137 D137M 0.23 0.34 0.21 0.72 1.06 137 D137N 2.26 0.75 0.52 1.03 1.13 137 D137P 0.41 1.22 0.48 1.03 1.23 137 D137Q 0.31 0.49 0.25 0.82 0.82 137 D137R 0.25 0.24 0.34 4.67 1.96 137 D137S 0.37 0.94 0.77 0.91 1.05 137 D137T 0.55 0.85 0.47 1.01 1.10 137 D137V 0.24 0.27 0.15 1.14 1.33 137 D137W 0.34 0.42 0.15 0.99 0.72 137 D137Y 0.36 0.50 0.28 0.83 0.91 138 T138A 3.42 1.04 1.06 1.05 1.26 138 T138C 2.40 0.97 0.95 1.03 1.30 138 T138D 0.23 0.11

0.76 1.68 138 T138E 2.03 0.49 0.56 0.78 1.58 138 T138F 0.64 0.53 0.37 1.42 4.28 138 T138G 0.51 0.75 0.63 0.99 0.94 138 T138H 0.58 0.51 0.47 1.14 1.06 138 T138I 0.54 0.86 0.75 0.95 0.99 138 T138K 0.40 0.39 0.18 0.86 1.68 138 T138L 1.26 1.01 0.90 1.01 1.19 138 T138M 0.75 0.97 0.97 1.09 1.14 138 T138N 0.51 0.52 0.43 2.14 2.93 138 T138P 0.40 0.66 0.71 1.23 1.63 138 T138Q 0.91 0.98 0.85 0.96 1.01 138 T138R 0.40 0.67 0.57 0.71 1.00 138 T138S 1.29 0.95 1.05 1.13 1.25 138 T138V 0.99 0.98 0.74 1.04 1.20 138 T138W 0.67 0.81 0.83 0.95 0.84 138 T138Y 0.34 0.83 0.54 1.37 1.26 139 L139A 0.40 1.16 0.39 0.97 0.82 139 L139C 1.15 1.04 0.89 0.95 1.04 139 L139D 1.03 0.94 0.95 0.98 1.07 139 L139E 0.17 0.74 0.78 1.07 0.99 139 L139F 0.46 0.77 0.48 1.04 1.25 139 L139G 0.60 1.04 0.95 1.07 1.06 139 L139H 0.45 0.40 0.50 0.73 0.94 139 L139I 0.19 0.87 0.93 0.89 0.98 139 L139K 0.65 1.03 0.97 0.97 1.10 139 L139M 0.29 0.77 0.76 0.88 0.43 139 L139N 0.30 0.94 0.85 0.97 1.02 139 L139P 0.18 0.19 0.29 40.66  1.02 139 L139Q 0.76 0.96 0.98 1.05 1.25 139 L139R 0.51 0.98 0.68 1.00 0.96 139 L139S 0.46 1.19 1.02 1.09 1.07 139 L139T 0.37 1.32 1.05 1.00 1.13 139 L139V 0.73 1.04 0.64 0.95 1.05 139 L139W 0.50 0.85 0.69 1.05 0.84 139 L139Y 1.51 1.00 0.50 2.68 1.87 140 D140A 0.98 1.14 1.00 1.01 1.09 140 D140C 0.24 0.71 0.75 0.73 0.90 140 D140E 0.47 0.87 0.63 0.85 0.87 140 D140F 0.38 0.75 0.45 0.85 0.73 140 D140G 0.25 0.87 0.63 0.72 0.72 140 D140H 0.26 0.77 0.72 0.86 0.94 140 D140I 0.45 0.96 0.84 1.09 1.05 140 D140K 0.48 0.75 0.62 0.77 1.01 140 D140L 0.28 0.80 0.81 0.95 0.91 140 D140M 0.34 1.08 0.89 1.08 1.00 140 D140N 0.29 0.58 0.37 0.81 1.02 140 D140P 0.10 0.14 0.17 10.60  12.72  140 D140Q 1.14 1.06 0.94 1.06 1.10 140 D140R 0.36 0.75 0.47 1.09 1.02 140 D140S 0.65 1.06 0.92 1.04 1.05 140 D140T 0.16 0.34 0.20 1.64 1.96 140 D140V 0.24 0.85 0.55 0.88 1.11 140 D140W 0.94 0.98 0.92 0.98 1.09 140 D140Y 0.34 0.78 0.64 0.91 1.01 141 K141A 0.83 0.94 0.54 0.54 141 K141C 1.16 1.10 0.56 0.51 141 K141D 0.72 0.61 1.39 0.68 141 K141E 0.65 0.29 2.54 2.73 141 K141F 0.52 0.51 2.02 1.67 141 K141G 0.49 0.58 1.62 1.45 141 K141H 1.32 1.17 0.67 0.83 141 K141I 1.18 0.57 1.58 1.16 141 K141L 0.87 0.35 2.74 1.77 141 K141M 0.45 0.51 1.93 1.54 141 K141N 0.54 0.60 2.08 1.22 141 K141R 0.63 1.11 1.14 0.32 141 K141S 0.81 0.72 1.14 1.63 141 K141T 0.45 0.60 2.03 1.72 141 K141V 0.40 0.46 2.92 2.99 141 K141Y 0.65 0.07 1.05 3.34 143 P143A 0.46 1.10 0.93 1.04 0.83 143 P143C 2.28 0.75 0.82 0.68 0.66 143 P143D 0.29 0.30 0.14 5.14 4.04 143 P143E 1.87 0.96 0.98 1.05 1.14 143 P143F 0.57 1.29 0.70 0.98 0.90 143 P143G 0.67 0.71 1.06 0.92 1.04 143 P143H 0.49 0.92 0.76 0.97 0.97 143 P143I 0.86 1.06 1.03 1.02 1.10 143 P143K 0.30 0.78 0.93 0.93 0.79 143 P143L 1.12 0.74 0.84 1.08 1.09 143 P143M 0.18 0.06 0.14 3.72 1.81 143 P143N 0.56 1.16 1.03 1.06 0.91 143 P143Q 1.57 1.09 0.90 1.09 1.16 143 P143R 0.36 0.77 0.97 0.99 0.77 143 P143S 1.92 1.11 1.05 1.06 1.25 143 P143T 0.49 1.33 0.96 0.86 0.85 143 P143V 0.51 0.73 0.68 0.81 0.90 143 P143W 1.09 0.91 1.02 0.95 0.99 143 P143Y 1.06 0.82 0.70 0.95 0.98 144 L144A 1.88 1.05 1.15 0.95 0.67 144 L144C 0.36 1.10 1.11 0.75 0.75 144 L144D 0.37 0.79 0.76 0.76 0.72 144 L144E 1.08 1.06 0.99 0.90 0.77 144 L144F 0.77 1.03 0.93 0.96 0.85 144 L144G 0.33 0.88 0.76 0.76 0.67 144 L144H 0.81 1.04 0.75 0.89 0.87 144 L144I 0.68 1.03 0.95 0.93 0.72 144 L144K 0.34 1.02 0.72 0.82 0.76 144 L144M 0.32 1.09 0.96 0.94 0.85 144 L144N 0.44 0.81 0.59 0.86 0.81 144 L144P 0.26 0.50 0.16 0.71 1.07 144 L144Q 0.36 1.00 0.77 1.00 1.31 144 L144S 0.35 0.19 0.24 1.57 2.43 144 L144T 0.27 0.93 0.63 0.74 0.78 144 L144V 0.32 0.83 0.70 0.76 0.77 144 L144W 0.33 1.15 1.00 0.84 0.98 144 L144Y 1.02 1.05 0.85 0.99 0.96 147 Q147A 0.09 0.94 0.82 0.91 0.87 147 Q147C 0.14 0.68 0.48 0.92 0.81 147 Q147E 0.86 1.00 0.88 1.07 1.05 147 Q147F 0.10 0.65 0.66 1.03 1.11 147 Q147G 0.39 1.01 0.93 1.02 1.00 147 Q147I 0.14 0.88 0.54 0.96 0.75 147 Q147K 0.13 0.85 0.61 0.87 0.89 147 Q147L 0.11 0.77 0.47 1.08 0.91 147 Q147M 0.23 0.85 0.56 0.75 0.69 147 Q147N 0.67 1.04 0.83 0.99 0.97 147 Q147P 0.20 0.25 0.26 0.66 1.19 147 Q147R 0.46 1.01 0.80 0.97 0.85 147 Q147S 0.63 0.97 0.79 1.12 1.02 147 Q147T 0.83 0.97 0.85 1.10 1.09 147 Q147V 0.19 0.71 0.59 0.89 0.94 147 Q147W 0.20 0.61 0.63 0.74 0.82 150 A150C 0.15 0.40 0.42 0.61 0.90 150 A150D 0.71 0.82 0.92 0.98 0.98 150 A150E 0.37 1.09 0.90 1.05 1.03 150 A150F 0.33 1.10 0.87 1.01 0.93 150 A150G 0.72 0.95 0.87 1.11 1.07 150 A150H 1.02 1.02 0.92 1.14 1.21 150 A150I 0.28 0.83 0.92 1.03 1.05 150 A150K 0.66 1.00 0.87 1.07 1.07 150 A150L 0.46 0.59 0.45 1.12 1.92 150 A150M 0.51 0.80 0.70 1.05 1.05 150 A150N 0.82 0.98 0.81 1.09 1.14 150 A150P 0.14 0.44 0.42 1.18 1.65 150 A150Q 0.64 1.02 0.86 1.17 1.15 150 A150R 0.36 0.91 0.87 0.94 1.05 150 A150S 0.53 1.05 0.95 0.99 1.05 150 A150T 0.13 0.96 0.67 0.99 0.83 150 A150V 0.35 0.81 0.87 0.99 0.98 150 A150W 0.61 0.82 0.77 1.01 0.99 150 A150Y 0.45 0.54 0.49 0.74 0.76 153 R153A 0.23 0.50 0.72 0.63 0.64 153 R153C 0.26 0.51 0.86 0.94 0.88 153 R153D 0.35 0.35 0.54 0.75 0.72 153 R153E 0.27 0.69 0.75 0.79 0.80 153 R153F 0.21 0.38 0.69 0.68 0.58 153 R153G 0.21 0.31 0.59 0.62 0.92 153 R153H 0.39 0.71 0.79 0.97 0.97 153 R153I 0.22 0.28 0.37 0.63 1.09 153 R153K 0.26 0.39 0.54 0.78 0.92 153 R153L 0.19 0.30 0.40 0.94 0.44 153 R153M 0.31 0.70 0.86 0.92 1.05 153 R153N 0.22 0.20 0.42 1.12 1.23 153 R153P 0.23 0.14 0.10 0.39 2.17 153 R153Q 0.24 0.52 0.99 0.77 1.05 153 R153S 0.41 0.78 0.99 0.93 0.93 153 R153T 0.31 0.63 0.74 0.84 0.94 153 R153V 0.23 0.28 0.59 0.63 0.81 153 R153W 0.42 0.77 1.07 0.90 0.87 153 R153Y 0.32 0.60 0.73 0.84 0.90 154 T154A 0.73 1.06 0.86 0.98 1.00 154 T154C 0.05 0.96 0.94 1.36 1.39 154 T154D 0.64 0.68 0.82 1.26 1.90 154 T154E 0.35 0.83 0.65 0.96 0.91 154 T154F 0.21 0.88 0.86 0.94 0.84 154 T154G 0.61 0.92 0.94 0.99 0.94 154 T154H 0.46 0.97 0.86 1.03 1.02 154 T154I 0.43 0.84 0.69 0.94 0.94 154 T154K 0.43 0.93 0.85 0.98 0.89 154 T154L 0.34 0.70 0.67 0.98 0.89 154 T154M 0.32 0.90 0.77 1.08 1.05 154 T154N 0.05 0.23 0.25 2.37 3.90 154 T154P 0.30 0.46 0.34 0.78 0.37 154 T154Q 0.17 0.71 1.01 0.87 0.93 154 T154R 0.40 0.96 1.10 1.04 0.95 154 T154S 0.79 1.03 0.91 1.05 1.06 154 T154V 0.33 0.86 0.99 1.01 0.96 154 T154W 0.43 0.97 0.98 0.93 0.94 154 T154Y 0.59 0.80 0.80 0.78 1.13 157 K157A 0.68 1.04 0.93 1.01 0.91 157 K157D 0.69 0.96 1.01 1.12 0.90 157 K157E 0.10 0.92 0.75 1.03 0.73 157 K157F 0.40 1.01 0.94 1.07 0.95 157 K157G 0.50 0.98 0.76 1.04 0.93 157 K157H 0.46 1.01 0.85 1.08 0.94 157 K157I 0.71 1.00 0.81 1.05 0.94 157 K157L 0.18 0.88 0.60 1.04 0.75 157 K157M 0.33 0.99 0.77 0.99 0.88 157 K157P 0.11 0.57 0.63 3.04 1.86 157 K157Q 0.50 1.00 0.70 1.00 0.88 157 K157R 0.82 1.02 1.19 0.98 0.92 157 K157T 0.14 0.79 0.47 1.02 0.83 157 K157V 0.46 0.99 0.86 0.99 0.91 157 K157W 1.06 0.95 0.84 1.04 0.93 157 K157Y 0.77 0.98 0.92 1.00 0.84 158 N158A 0.43 0.95 0.80 0.94 1.03 158 N158C 0.25 0.77 0.68 1.03 0.92 158 N158D 0.55 0.99 0.86 1.01 0.90 158 N158E 0.54 0.93 0.88 0.94 1.18 158 N158F 0.36 0.90 0.70 1.02 0.89 158 N158G 0.24 1.06 0.66 0.89 0.75 158 N158H 0.43 0.93 0.72 0.98 0.89 158 N158I 0.92 1.06 0.91 1.13 1.09 158 N158L 0.62 1.12 0.81 0.97 0.99 158 N158M 0.19 0.38 0.27 0.40 0.88 158 N158P 0.19 0.81 0.43 0.85 1.12 158 N158Q 0.41 1.05 0.95 0.93 1.06 158 N158R 0.23 0.74 0.43 0.76 0.74 158 N158S 0.61 0.92 0.61 0.88 0.84 158 N158T 0.18 0.63 0.85 0.80 2.47 158 N158V 0.65 0.92 0.75 1.00 0.87 158 N158W 0.28 0.90 0.65 0.80 0.73 158 N158Y 0.19 0.90 0.92

0.32 161 N161A 0.39 1.43 1.91 0.99 0.97 161 N161C 0.62 0.95 1.12 1.05 1.06 161 N161E 0.76 1.03 1.07 1.12 1.12 161 N161F 0.62 1.07 1.07 1.01 1.05 161 N161G 0.55 0.99 1.02 0.99 0.95 161 N161H 0.55 0.92 0.83 1.04 1.02 161 N161I 0.51 0.89 0.68 1.03 1.01 161 N161K 0.43 0.84 0.67 1.03 1.20 161 N161L 0.32 0.59 0.92 2.18 1.90 161 N161M 0.53 1.01 1.11 0.98 1.09 161 N161P 1.06 1.03 0.98 1.12 1.24 161 N161Q 0.68 1.02 0.97 1.04 1.07 161 N161R 0.40 0.75 0.34 0.41 0.44 161 N161S 0.58 1.01 1.06 0.90 0.90 161 N161T 1.06 1.05 0.96 1.01 1.18 161 N161V 0.59 1.00 0.97 1.00 1.12 161 N161W 0.46 0.91 1.31 1.02 0.95 161 N161Y 0.83 1.03 1.09 1.04 1.05 162 Y162A 0.15 0.78 0.73 0.97 1.11 162 Y162C 0.15 0.64 0.50 0.73 1.57 162 Y162D 0.11 0.39 0.61 1.14 1.90 162 Y162E 0.14 0.28 0.37 1.44 2.01 162 Y162F 0.76 1.04 0.87 1.11 1.03 162 Y162G 0.29 1.05 0.87 0.97 0.84 162 Y162H 0.61 1.04 0.91 1.12 1.16 162 Y162I 0.21 0.77 0.85 0.91 0.66 162 Y162K 0.16 0.83 0.93 1.06 0.89 162 Y162L 0.17 0.59 0.52 0.78 0.83 162 Y162M 0.17 0.66 0.61 0.79 0.81 162 Y162N 0.28 0.92 1.02 1.04 0.99 162 Y162P 0.14 0.33 0.21 0.95 1.01 162 Y162Q 0.18 0.93 0.81 0.86 0.75 162 Y162R 0.20 0.82 0.86 1.04 0.91 162 Y162S 0.20 1.05 0.67 1.01 0.91 162 Y162T 0.14 0.52 0.33 0.63 0.96 162 Y162V 0.20 0.67 0.89 1.02 0.87 162 Y162W 0.54 1.03 0.77 0.92 0.81 177 A177C 0.27 0.42 0.53 1.74 1.01 177 A177D 2.02 0.95 0.98 0.93 1.04 177 A177E 0.36 0.59 0.67 1.03 1.02 177 A177F 0.21 0.48 0.31 0.87 0.69 177 A177G 0.80 0.73 0.66 0.95 1.11 177 A177H 0.12 0.32 0.30 0.99 4.79 177 A177I 0.25 0.35 0.23 0.68 0.79 177 A177K 0.52 0.76 0.43 1.02 1.07 177 A177L 0.33 0.26 0.27 0.89 1.00 177 A177M 0.36 0.66 0.64 0.82 0.91 177 A177N 0.66 0.92 0.92 1.09 1.32 177 A177P 0.51 0.52 0.36 0.70 0.61 177 A177Q 0.21 0.46 0.42 1.40 1.12 177 A177R 0.25 0.54 0.42 1.04 1.04 177 A177S 1.45 1.01 0.84 1.06 1.31 177 A177T 0.45 0.62 0.46 0.86 0.74 177 A177V 0.30 0.58 0.42 0.78 0.63 177 A177W 0.37 0.56 0.28 1.22 0.85 177 A177Y 0.84 0.76 0.53 0.95 1.04 178 A178C 0.26 0.31 0.17 0.89 1.15 178 A178D 1.60 0.63 0.68 0.96 1.03 178 A178E 0.92 0.61 0.44 1.03 1.23 178 A178F 0.56 0.40 0.23 0.93 0.91 178 A178G 1.18 0.73 0.61 0.84 0.81 178 A178H 0.11 0.71 0.33 1.21 1.02 178 A178I 0.40 0.27 0.15 1.50 1.40 178 A178K 0.76 0.10 0.12 1.15 2.62 178 A178L 0.29 0.25 0.23 1.61 1.99 178 A178M 0.69 0.40 0.37 1.23 1.43 178 A178N 1.80 0.36 0.48 0.95 1.39 178 A178P 0.11 0.18 0.14 2.14 1.65 178 A178Q 1.22 0.51 0.49 1.17 1.36 178 A178R 0.43 0.19 0.23 1.45 1.85 178 A178S 0.35 0.80 0.62 1.05 0.93 178 A178T 0.21 0.39 0.30 2.08 1.42 178 A178V 1.07 0.46 0.36 1.10 1.21 178 A178W 0.88 0.16

1.65 2.37 178 A178Y 0.73 0.44

1.02 1.30 179 L179A 1.02 1.03 1.26 1.04 0.97 179 L179C 1.06 0.87 1.01 1.09 1.04 179 L179D 0.29 0.75 0.43 0.93 0.84 179 L179E 0.14 0.16 0.32 0.59 0.79 179 L179F 0.27 0.77 0.42 1.01 0.93 179 L179G 1.02 0.98 0.93 1.05 0.98 179 L179H 0.95 0.95 0.61 1.10 0.93 179 L179K 0.76 0.89 0.70 0.99 0.87 179 L179M 0.30 0.82 0.61 1.12 1.11 179 L179N 0.94 0.76 0.75 1.11 0.99 179 L179P 0.24 0.32 0.26 0.91 0.85 179 L179Q 0.34 0.85 1.03 1.18 1.19 179 L179R 0.13 0.17

0.27 0.92 179 L179S 0.78 0.95 1.02 1.08 1.10 179 L179T 0.79 0.90 0.86 1.11 1.00 179 L179V 0.82 0.96 0.79 1.06 1.03 179 L179W 0.40 0.94 0.54 0.85 0.71 179 L179Y 0.24 0.67 0.56 0.79 0.73 180 A180C 0.50 0.80 0.11 0.78 0.57 180 A180D 0.66 0.21 0.09 0.61 0.10 180 A180E 1.04 0.53 0.24 0.76 0.74 180 A180F 0.72 0.15

0.38 1.72 180 A180G 0.35 0.68 0.22 0.75 0.58 180 A180H 0.58 0.11 0.07 0.56 0.25 180 A180I 0.62 0.26 0.09 0.64 0.86 180 A180K 0.88 0.12

0.57 0.55 180 A180L 0.82 0.20 0.08 0.59 0.83 180 A180M 0.94 0.15

13.63  0.81 180 A180N 0.08 0.10 0.26

4.24 180 A180P 0.50 0.53 0.10 0.72 0.59 180 A180Q 0.85 0.35 0.13 0.58 0.58 180 A180R 0.69 0.05 0.11 4.95 3.56 180 A180S 1.32 0.89 0.77 1.04 1.01 180 A180T 0.76 0.44 0.18 0.60 0.55 180 A180V 0.32 0.19 0.23 0.85 0.84 180 A180W 1.14 0.09

0.65 1.85 180 A180Y 0.63 0.09

2.52 1.23 181 S181A 0.56 0.39 0.35 0.81 0.73 181 S181C 0.84 0.12 0.08 1.86 1.46 181 S181D 0.84 0.12 0.16 1.32 0.90 181 S181E 0.54 0.11

0.96 1.23 181 S181F 0.54 0.10

1.34 1.42 181 S181G 0.90 0.42 0.18 0.89 0.84 181 S181K 0.53 0.06

1.81 4.72 181 S181L 0.34 0.15

0.99 1.53 181 S181M 0.45 0.11 0.13 0.55 2.09 181 S181N 0.71 0.17 0.08 1.30 0.58 181 S181P 0.57 0.11 0.05 1.37 0.79 181 S181Q 0.42 0.11 0.08 2.41 1.67 181 S181R 0.58 0.07

0.18 1.33 181 S181T 1.01 0.17 0.09 1.53 1.19 181 S181V 0.58 0.13 0.09 1.76 1.08 181 S181W 0.76 0.05

0.96 3.23 181 S181Y 0.64 0.61 0.36 0.71 0.88 182 N182A 0.56 1.21 0.96 0.92 1.05 182 N182C 1.24 0.96 0.80 0.91 0.89 182 N182D 1.58 1.02 0.89 0.93 0.87 182 N182E 0.21 0.17

0.63 1.74 182 N182G 0.93 0.86 0.83 1.21 1.26 182 N182H 1.45 1.06 0.65 0.87 0.85 182 N182I 0.94 0.59 0.54 1.06 1.13 182 N182K 0.99 0.59 0.43 0.87 0.80 182 N182L 0.78 0.98 0.78 1.01 1.13 182 N182M 1.74 0.90 0.84 1.12 1.28 182 N182P 0.87 0.63 0.50 0.85 0.85 182 N182Q 0.18 0.13

1.70 1.48 182 N182R 0.69 0.73 0.46 1.03 1.03 182 N182S 1.09 1.07 0.98 0.91 0.92 182 N182T 1.06 0.95 0.89 0.94 0.99 182 N182V 1.10 0.74 0.59 0.76 0.77 182 N182W 2.82 1.06 1.36 1.10 1.26 182 N182Y 1.20 0.87 0.90 1.03 1.15 185 Y185A 0.07

0.29 2.19 1.88 185 Y185C 0.14 0.18 0.21

0.13 185 Y185D 0.17 0.08 0.16 0.55 1.10 185 Y185E 0.59 0.08 0.12 0.09 1.45 185 Y185F 0.86 1.08 0.91 1.06 1.05 185 Y185G 0.15 0.12 0.06

0.22 185 Y185H 0.59 0.88 0.34 0.64 0.46 185 Y185I 0.21 0.68 0.54 0.66 0.64 185 Y185L 1.88 1.19 1.11 1.02 1.09 185 Y185M 0.35 0.74 0.41 0.97 0.78 185 Y185P 0.22 0.10 0.20 0.33 0.89 185 Y185Q 0.12 0.22 0.14

0.62 185 Y185R 0.12 0.08 0.05 3.65 4.42 185 Y185S 0.16 0.41 0.16 0.32 0.26 185 Y185T 0.15 0.35 0.25 0.19 0.38 185 Y185V 0.24 0.16 0.15 0.60 0.37 185 Y185W 0.28 0.80 0.77 0.83 0.97 186 S186A 0.87 0.88 0.89 0.96 1.06 186 S186C 0.27 0.64 0.67 1.22 2.83 186 S186D 1.73 0.97 0.96 1.04 1.19 186 S186E 2.57 0.82 0.72 1.06 1.19 186 S186F 0.33 0.64 0.48 1.42 1.61 186 S186G 0.49 1.02 0.59 1.01 1.09 186 S186H 2.18 0.96 0.70 1.09 1.40 186 S186I 3.18 0.88 0.86 1.09 1.27 186 S186K 1.22 0.95 0.61 1.03 1.00 186 S186L 1.03 1.06 0.92 1.13 1.31 186 S186M 1.21 0.88 0.73 1.06 1.27 186 S186N 0.16 0.65 0.43 0.91 1.04 186 S186P 0.19 0.78 0.22 0.77 0.82 186 S186Q 0.69 1.10 0.95 1.07 1.21 186 S186R 2.78 0.90 0.73 0.89 1.07 186 S186T 0.48 1.08 0.87 1.14 1.21 186 S186V 0.92 1.03 0.91 1.03 1.26 186 S186W 0.64 0.94 0.61 1.01 1.14 186 S186Y 0.21 0.24

2.68 4.96 188 A188C 0.67 0.57 0.62 0.99 1.05 188 A188D 0.74 0.55 0.69 1.10 1.38 188 A188E 0.42 0.79 0.62 1.41 1.06 188 A188F 0.48 0.88 0.50 0.93 0.80 188 A188H 0.79 0.64 0.60 1.32 1.39 188 A188I 0.41 0.52 0.37 1.60 1.05 188 A188K 0.72 0.43 0.31 0.79 0.58 188 A188L 0.82 0.63 0.53 1.41 0.92 188 A188M 0.39 0.60 0.52 3.51 3.70 188 A188N 0.33 0.39 0.42 2.28 1.14 188 A188P 0.36 1.06 0.82 1.01 0.89 188 A188Q 0.12 0.85 0.72 0.88 0.79 188 A188R 0.36 0.87 0.49 0.93 0.83 188 A188S 0.63 1.21 1.17 1.05 1.00 188 A188T 1.02 0.67 0.60 1.24 1.29 188 A188V 0.55 0.92 0.63 0.96 0.94 188 A188W 0.53 0.61 0.57 0.90 1.02 188 A188Y 0.54 0.45 0.30 1.00 0.98 189 D189A 0.29 0.77 0.73 0.79 0.69 189 D189C 0.38 0.89 0.82 0.90 0.76 189 D189E 0.44 0.98 0.84 0.94 0.89 189 D189F 0.39 0.82 0.64 0.94 0.99 189 D189G 0.27 0.87 0.86 0.88 0.71 189 D189H 0.39 0.91 0.52 0.91 0.83 189 D189I 0.16 0.52 0.39 0.63 0.61 189 D189K 0.37 0.96 0.78 0.89 0.73 189 D189L 0.33 1.04 0.82 0.89 0.85 189 D189N 0.55 0.95 1.05 1.00 0.96 189 D189P 0.12 0.30 0.22 0.72 0.76 189 D189Q 0.73 0.98 0.72 0.93 0.86 189 D189R 0.38 0.96 0.82 0.94 0.88 189 D189S 0.14 0.60 0.32 0.86 1.22 189 D189T 0.21 0.84 0.84 0.85 0.76 189 D189V 0.36 1.03 0.99 1.03 0.91 189 D189W 0.13 0.18 0.34 0.45 1.06 189 D189Y 0.32 0.52 0.46 1.30 1.34 190 G190A 0.79 0.81 0.62 0.86 0.77 190 G190C 0.35 0.80 0.87 0.78 0.83 190 G190D 0.54 0.95 0.82 0.93 0.92 190 G190E 0.18 0.71 0.49 0.89 1.05 190 G190H 0.11 0.40 0.24 0.35 0.80 190 G190I 0.13 0.45 0.39 0.49 0.55 190 G190K 0.27 0.87 0.58 0.91 0.94 190 G190L 0.74 0.89 0.60 0.82 1.04 190 G190M 0.45 0.58 0.45 0.93 1.39 190 G190N 0.56 1.00 0.73 0.93 0.98 190 G190P 0.14 0.09 0.26 26.00  3.19 190 G190Q 0.59 1.04 0.80 0.95 1.04 190 G190R 0.49 0.74 0.44 0.74 1.10 190 G190S 0.86 1.15 0.96 0.99 1.13 190 G190T 0.50 0.84 0.79 1.06 1.15 190 G190V 0.25 0.90 0.57 0.88 0.83 190 G190W 0.53 0.79 0.42 0.90 0.89 190 G190Y 0.38 0.95 0.83 0.84 0.91 191 G191C 0.14 0.17 0.20 0.82 1.34 191 G191D 0.20 0.39 0.27 0.28 0.13 191 G191E 0.15 0.22 0.10 0.75 0.79 191 G191F 0.17 0.19 0.25 0.51 0.94 191 G191I 0.14 0.18 0.22 0.55 0.95 191 G191L 0.15 0.24 0.18 0.95 0.91 191 G191M 0.13 0.23 0.40 0.46 0.88 191 G191P 0.12 0.19 0.29 0.56 1.20 191 G191Q 0.19 0.65 0.51 0.89 0.74 191 G191R 0.13 0.21 0.24 0.62 1.52 191 G191S 0.09 0.38 0.05 0.75 1.30 191 G191T 0.12 0.23 0.10 0.47 1.18 191 G191V 0.13 0.21 0.17 0.51 1.34 191 G191W 0.12 0.18 0.12 0.07 1.08 192 V192A 0.42 0.95 0.73 1.00 0.80 192 V192C 0.87 0.68 0.52 0.88 0.73 192 V192D 0.38 0.39 0.34 0.48 0.50 192 V192E 0.69 1.02 0.82 0.98 0.98 192 V192F 0.20 0.19 0.06

0.08 192 V192G 0.16 0.46 0.21 0.47 0.53 192 V192H 0.30 0.75 0.40 0.83 0.86 192 V192K 0.34 0.58 0.32 0.77 0.72 192 V192L 0.76 1.04 0.55 1.03 1.00 192 V192M 0.46 0.96 0.44 1.01 0.95 192 V192P 0.35 0.80 0.29 0.93 0.83 192 V192R 0.60 1.01 0.76 1.02 0.99 192 V192S 0.29 0.76 0.46 0.93 0.85 192 V192T 0.34 0.47 0.22 0.75 0.87 192 V192W 0.25 0.53 0.25 0.72 0.65 192 V192Y 0.25 0.64 0.25 0.81 0.75 193 A193C 0.31 0.99 0.93 0.94 0.71 193 A193D 0.49 1.07 1.02 1.11 0.90 193 A193E 0.31 1.05 0.89 1.01 0.72 193 A193F 0.35 1.06 0.75 0.99 0.80 193 A193G 0.17 0.68 0.41 1.32 1.04 193 A193I 0.37 1.12 0.88 0.97 0.82 193 A193K 0.32 0.85 0.71 0.99 0.85 193 A193L 0.31 1.10 0.88 0.90 0.81 193 A193M 0.44 1.12 0.82 0.94 0.90 193 A193N 0.74 0.96 0.84 1.05 0.97 193 A193P 0.23 0.99 0.79 0.95 0.79 193 A193Q 0.48 1.11 0.82 1.02 0.96 193 A193R 0.37 1.06 0.73 0.98 0.89 193 A193S 0.48 0.98 0.81 0.95 0.85 193 A193T 0.10 0.40 0.31 1.16 0.77 193 A193V 0.45 1.06 0.85 0.99 0.87 193 A193W 0.49 1.11 1.05 0.98 0.87 193 A193Y 0.40 0.78 0.63 0.88 0.79 194 K194A 0.30 0.67 0.86 0.95 1.00 194 K194C 0.22 1.08 1.72 0.88 0.88 194 K194D 0.61 0.98 1.07 1.00 1.03 194 K194E 0.44 1.00 1.14 1.02 1.14 194 K194F 0.67 1.03 0.94 1.04 1.13 194 K194G 0.43 0.92 0.65 0.92 0.96 194 K194H 0.60 0.99 0.92 1.05 1.15 194 K194I 0.24 0.38 0.49 0.50 0.59 194 K194L 0.61 0.98 0.90 1.01 1.22 194 K194M 0.61 1.02 0.86 1.03 1.17 194 K194N 0.54 0.99 1.02 1.04 1.17 194 K194P 0.32 0.39 0.55 0.46 0.38 194 K194Q 0.64 1.00 1.00 1.03 1.17 194 K194R 0.66 1.04 0.88 1.01 1.23 194 K194S 0.66 1.00 0.83 1.12 1.03 194 K194T 0.08 0.11 1.09 1.17 9.80 194 K194V 0.23 0.30 0.71 0.55 0.62 194 K194W 0.43 0.93 0.98 0.98 1.01 194 K194Y 0.08 0.11 0.36 0.60 2.65 196 K196A 0.43 0.98 0.81 0.91 0.85 196 K196F 0.52 1.02 0.93 1.02 1.04 196 K196G 0.13 0.71 0.30 0.96 0.86 196 K196H 0.26 1.02 0.77 1.00 1.03 196 K196L 0.63 0.95 0.71 1.00 1.00 196 K196M 0.84 0.98 0.94 0.97 1.02 196 K196Q 0.67 0.98 0.74 0.97 1.05 196 K196W 0.31 0.84 0.51 0.92 0.86 196 K196Y 0.07 1.23 0.50 0.94 0.96 197 N197A 0.56 1.22 1.12 0.98 0.96 197 N197C 0.30 0.98 0.72 1.09 1.02 197 N197D 1.50 1.12 1.08 1.06 1.07 197 N197F 0.29 0.99 0.69 0.91 0.85 197 N197G 1.53 1.10 0.92 1.07 1.10 197 N197H 1.02 1.07 0.80 1.08 1.10 197 N197I 0.60 1.10 0.74 1.03 1.02 197 N197K 0.45 1.00 0.48 0.89 0.94 197 N197L 0.29 0.97 0.60 0.97 0.95 197 N197M 0.53 1.14 0.82 1.05 1.05 197 N197P 0.41 1.01 0.79 0.93 0.93 197 N197Q 0.39 0.95 0.72 0.98 0.98 197 N197R 0.26 0.98 0.74 0.93 1.05 197 N197T 0.68 1.07 0.78 1.04 1.00 197 N197V 0.67 1.12 0.87 0.97 1.04 197 N197W 0.46 0.99 0.77 0.98 1.01 197 N197Y 0.18 0.56 0.06 0.63 1.17 201 T201A 0.54 0.61 0.61 0.87 0.83 201 T201C 0.34 0.79 0.62 0.92 0.99 201 T201D 0.30 1.00 1.08 0.89 0.73 201 T201E 0.18 0.84 0.43 0.87 0.71 201 T201F 0.32 0.81 0.61 0.85 0.89 201 T201G 0.33 1.06 0.93 0.90 0.86 201 T201H 0.33 0.96 0.75 0.86 0.86 201 T201I 0.20 0.86 0.58 0.89 0.70 201 T201K 0.25 1.12 0.87 0.87 0.74 201 T201L 0.40 0.98 0.63 0.93 0.89 201 T201M 0.43 0.97 0.66 0.90 0.95 201 T201N 0.29 0.85 0.66 0.85 0.85 201 T201P 0.22 0.63 0.42 0.94 1.41 201 T201Q 0.42 0.95 0.70 0.86 0.95 201 T201R 0.33 0.56 0.39 1.28 1.26 201 T201S 0.38 1.01 0.70 0.90 0.83 201 T201V 0.28 0.56 0.53 1.07 1.11 201 T201W 0.23 0.66 0.50 0.92 0.93 201 T201Y 0.24 0.55 0.40 1.05 2.18 203 R203A 0.71 0.93 0.75 0.69 0.88 203 R203F 0.85 0.92 0.99 0.67 0.87 203 R203G 0.60 1.07 1.13 0.66 1.39 203 R203H 0.81 1.04 1.24 2.57 3.09 203 R203I 0.39 0.70 0.64 1.46 3.02 203 R203K 0.96 0.84 0.99 0.55 0.75 203 R203L 0.46 1.14 0.54 1.08 1.24 203 R203M 0.58 0.88 0.91 0.99 1.25 203 R203N 0.36 1.08 0.82 1.06 1.20 203 R203P 0.29 0.49 0.28 2.85 4.58 203 R203Q 0.80 0.99 0.78 0.84 0.86 203 R203S 0.54 1.19 0.53 1.99 1.96 203 R203T 0.66 1.04 0.82 0.99 0.91 203 R203V 0.85 0.94 0.89 0.73 0.78 203 R203W 0.39 1.10 0.63 2.28 2.42 203 R203Y 0.57 1.09 1.03 1.03 1.18 204 Q204A 0.91 0.98 0.98 0.99 0.99 204 Q204C 1.03 1.13 0.91 1.00 0.89 204 Q204D 1.23 1.04 0.73 1.00 1.00 204 Q204E 1.08 1.12 1.05 0.93 1.04 204 Q204F 0.48 0.74 0.94 1.13 0.94 204 Q204G 1.14 1.08 0.87 0.77 0.75 204 Q204H 1.30 1.12 0.84 1.16 0.91 204 Q204I 0.86 0.68 0.88 1.33 1.57 204 Q204K 1.13 0.75 0.91 1.61 1.28 204 Q204L 0.45 0.82 0.81 0.98 0.96 204 Q204M 0.44 0.85 0.87 0.78 0.76 204 Q204N 0.59 0.96 0.96 1.04 0.83 204 Q204P 0.36 0.50 0.83 1.09 1.19 204 Q204R 0.95 0.87 0.89 1.36 1.06 204 Q204S 0.68 0.53 1.00 0.97 1.95 204 Q204T 1.94 1.08 0.93 1.07 1.18 204 Q204V 0.85 0.78 0.74 1.29 1.32 204 Q204W 0.13 0.07 0.22 0.11 0.15 206 V206C 0.46 0.83 0.43 1.52 1.48 206 V206D 0.38 0.24 0.10 3.69 7.75 206 V206E 0.35 0.45 0.39 1.54 3.44 206 V206F 0.76 1.00 0.74 2.06 2.29 206 V206G 0.33 0.94 0.79 1.56 1.54 206 V206I 0.35 1.56 0.83 1.97 1.61 206 V206L 0.82 1.44 0.87 0.70 0.84 206 V206M 0.42 1.41 0.49 1.75 1.64 206 V206P 0.48 0.24 0.30 3.72 7.12 206 V206Q 0.27 1.35 0.40 2.88 2.34 206 V206R 0.24 0.72 0.45 7.05 6.46 206 V206S 0.44 0.48 0.44 1.64 2.37 206 V206T 0.71 1.30 0.69 1.07 1.06 206 V206W 0.77 0.50 0.29 0.71 2.48 206 V206Y 0.66 1.19 0.57 1.17 1.58 207 V207A 0.72 1.09 0.93 0.94 1.01 207 V207C 0.69 1.07 0.83 0.94 0.99 207 V207D 0.11

1.44 1.05 207 V207E 0.56 1.04 0.70 0.79 1.01 207 V207F 0.78 1.11 0.67 0.87 1.09 207 V207G 0.83 0.98 0.90 1.12 1.01 207 V207I 0.69 1.06 0.84 0.94 1.03 207 V207K 0.75 0.97 0.67 1.03 1.04 207 V207L 0.64 1.01 0.73 0.70 1.09 207 V207M 0.58 0.92 0.62 1.00 1.03 207 V207N 0.68 0.84 0.53 1.01 1.03 207 V207P 0.20 0.10

1.83 2.90 207 V207Q 0.62 1.04 0.72 0.89 1.01 207 V207R 0.99 0.93 0.69 0.86 1.10 207 V207S 0.18

11.51  0.58 207 V207W 0.96 0.95 0.58 1.13 1.06 207 V207Y 0.78 0.95 0.62 1.06 1.01 210 S210A 0.69 0.94 0.87 0.94 0.91 210 S210E 0.28 0.80 0.64 0.85 0.76 210 S210F 0.43 0.80 0.53 0.92 0.81 210 S210G 0.39 0.70 0.49 0.94 0.83 210 S210H 0.32 0.90 0.80 0.93 0.72 210 S210I 0.78 0.72 0.56 0.91 0.84 210 S210L 0.06 1.45 1.58 0.93 0.86 210 S210N 0.45 0.93 1.02 0.93 0.92 210 S210P 0.31 0.78 0.80 0.86 0.77 210 S210R 0.38 0.91 0.62 0.94 0.88 210 S210T 0.55 0.95 0.76 0.96 0.89 210 S210V 0.33 0.82 0.65 0.99 0.93 210 S210W 0.38 1.02 0.97 1.00 0.89 210 S210Y 0.28 0.77 0.58 0.96 0.87 214 T214A 0.23 1.00 0.60 0.84 1.11 214 T214C 0.95 1.08 0.98 0.92 0.94 214 T214D 0.13 0.57 0.55 0.65 1.29 214 T214E 0.16 0.54 0.32 0.90 1.02 214 T214F 0.19 0.89 0.57 0.62 0.84 214 T214G 0.11 0.62 0.47 0.61 0.75 214 T214H 0.88 1.05 0.97 1.04 1.13 214 T214I 1.00 1.08 1.00 1.06 1.03 214 T214K 0.25 0.41 0.25 0.87 0.74 214 T214L 0.33 1.07 0.31 0.61 0.40 214 T214N 1.80 1.04 1.04 1.10 1.20 214 T214P 0.62 0.99 0.90 1.03 0.95 214 T214Q 0.86 1.05 0.78 1.03 1.08 214 T214R 0.37 0.17

0.48 1.29 214 T214S 0.54 1.03 0.86 0.94 1.08 214 T214V 1.01 1.13 1.03 1.01 0.97 214 T214W 0.32 0.29 0.32 0.87 1.07 214 T214Y 0.36 0.50 0.47 0.68 0.62 225 N225A 0.26 0.36 0.51 0.54 0.38 225 N225C 0.15 0.13 0.41 1.24 0.81 225 N225D 0.34 0.36 0.47 0.54 0.25 225 N225E 0.15 0.11 0.21 0.87 0.31 225 N225F 0.13 0.17 0.29 0.77 0.42 225 N225G 0.10 0.31 0.20 0.70 0.69 225 N225H 0.12 0.13 0.17 0.80 0.30 225 N225I 0.08 0.17 0.22 0.80 0.37 225 N225K 0.26 0.12 0.17 0.98 2.90 225 N225L 0.11 0.24 0.36 0.87 1.11 225 N225M 0.12 0.74 0.47 0.99 0.78 225 N225P 0.14 0.29 0.44 0.58 0.74 225 N225Q 0.14 0.44 0.25 0.60 0.41 225 N225R 0.11 0.13 0.21 1.69 1.02 225 N225S 0.14 0.40 0.45 0.68 0.82 225 N225T 0.21 0.50 0.34 0.56 0.76 225 N225V 0.20 0.38 0.30 0.84 0.33 225 N225W 0.20 0.13 0.20 1.87 1.45 225 N225Y 0.19 0.20 0.17 0.75 0.52 226 L226C 0.15 0.65 0.39 0.73 0.56 226 L226D 0.16 0.18 0.31 0.68 1.08 226 L226E 0.20 0.29 0.31 0.93 1.23 226 L226F 0.21 0.57 0.34 0.80 0.74 226 L226G 0.16 0.29 0.15 0.41 0.51 226 L226H 0.19 0.32 0.29 0.81 0.95 226 L226M 0.67 1.02 0.80 1.12 1.05 226 L226N 0.12 0.35 0.21 0.64 0.81 226 L226P 0.21 0.21 0.26 1.02 1.62 226 L226R 0.14 0.24 0.16 0.73 1.18 226 L226S 0.23 0.59 0.27 0.70 0.80 226 L226T 0.14 0.46 0.15 0.71 0.68 226 L226V 0.26 0.86 0.43 0.85 0.83 226 L226W 0.18 0.26 0.24 0.71 1.94 226 L226Y 0.15 0.28 0.35 0.53 1.17 228 T228A 0.22 0.48 0.87 0.29 0.49 228 T228C 0.12 0.37 0.79 0.49 0.81 228 T228F 0.22 0.14 0.23 0.92 1.09 228 T228G 0.25 0.46 0.67 0.32 0.44 228 T228H 0.14 0.24 0.73 0.46 1.00 228 T228I 0.23 0.23 0.36 0.76 0.88 228 T228K 0.19 0.11 0.31 0.54 1.35 228 T228M 0.28 0.34 0.41 0.55 0.62 228 T228N 0.23 0.49 0.28 0.54 0.49 228 T228P 0.14 0.25 0.42 0.26 0.78 228 T228R 0.38 0.11 0.74 0.90 1.70 228 T228S 0.22 0.63 0.41 0.55 0.52 228 T228V 0.56 0.69 0.13 0.90 0.67 228 T228W 0.18 0.08 0.42 9.87 33.17  228 T228Y 0.28 0.12 0.43

1.28 229 N229A 0.56 0.88 0.80 0.75 0.68 229 N229C 0.19 1.06 0.65 0.90 1.09 229 N229D 0.16 0.36

0.87 0.78 229 N229E 0.42 0.73 0.54 0.89 0.69 229 N229F 0.24 0.44 0.50 0.97 0.90 229 N229G 0.52 0.77 0.78 0.77 0.70 229 N229H 0.66 0.76 0.60 0.95 1.00 229 N229I 0.49 0.78 0.81 0.89 0.93 229 N229K 0.84 0.81 0.64 0.90 0.86 229 N229L 0.39 0.48 0.20 0.61 0.61 229 N229M 0.54 0.77 0.43 0.90 0.86 229 N229P 0.35 0.67 0.34 0.80 0.85 229 N229Q 0.17 0.48 0.33 0.94 1.72 229 N229R 0.42 0.81 0.53 0.93 0.81 229 N229S 0.49 0.92 0.69 0.84 0.72 229 N229T 0.36 0.67 0.53 0.64 0.60 229 N229V 0.48 0.56 0.44 0.74 0.90 229 N229W 0.33 0.64 0.60 0.75 0.73 229 N229Y 0.30 0.90 0.66 0.96 0.93 230 L230A 0.18 0.85 0.43 0.54 0.28 230 L230C 0.27 0.77 0.51 0.62 0.39 230 L230D 0.23 0.45 0.26 0.55 0.43 230 L230E 0.30 0.85 0.45 0.85 0.51 230 L230F 0.46 0.88 0.42 0.80 0.62 230 L230G 0.33 0.70 0.35 0.84 0.39 230 L230H 0.37 0.57 0.17 3.13 1.86 230 L230I 0.22 0.63 0.48 0.71 0.62 230 L230K 0.11 1.05 0.15 0.86 0.52 230 L230M 0.14 0.65 0.59 0.80 0.76 230 L230N 0.55 0.97 0.50 0.71 0.51 230 L230P 0.26 0.71 0.32 0.49 0.40 230 L230Q 0.30 0.78 0.37 0.79 0.51 230 L230R 0.87 1.03 0.42 0.92 0.79 230 L230S 0.36 0.63 0.43 1.04 0.66 230 L230T 0.42 1.04 0.52 0.78 0.67 230 L230V 0.29 0.82 0.36 0.72 0.62 230 L230W 0.14 0.28 0.16 0.80 0.81 230 L230Y 0.65 0.92 0.72 0.87 0.79 231 G231A 1.45 0.98 1.13 1.00 0.98 231 G231C 0.43 0.72 0.24 0.89 0.66 231 G231E 0.13 0.15 0.10 0.67 1.05 231 G231F 0.54 0.82 0.22 0.86 0.70 231 G231H 0.39 0.78 0.32 0.88 0.80 231 G231I 0.17 0.14 0.56 1.07 1.44 231 G231K 0.33 0.75 0.62 0.84 0.74 231 G231L 0.45 0.92 0.65 0.74 0.58 231 G231M 0.16 0.24 0.60 0.81 1.41 231 G231N 0.58 0.87 0.93 0.97 0.98 231 G231P 0.16 0.21 0.45 0.72 0.81 231 G231Q 0.29 0.13 0.22 0.77 1.25 231 G231R 0.33 0.43 0.36 0.79 0.79 231 G231S 0.81 1.01 0.62 1.01 0.96 231 G231T 0.96 0.66 0.44 0.89 0.68 231 G231V 0.19 0.55 0.55 0.54 0.42 231 G231W 0.73 0.80 0.16 0.91 0.74 231 G231Y 0.75 0.85 0.58 0.83 0.65 232 T232A 0.31 0.67 0.99 1.00 0.83 232 T232C 0.36 0.63 1.16 0.71 0.97 232 T232E 0.34 0.64 0.88 0.91 0.85 232 T232F 0.31 0.62 0.87 0.90 0.97 232 T232I 0.23 0.49 0.81 0.94 0.96 232 T232K 0.63 0.76 0.66 0.97 1.02 232 T232L 0.20 0.55 0.96 0.91 0.85 232 T232N 0.22 0.21 0.52 0.47 0.65 232 T232P 0.52 0.79 0.95 0.89 1.02 232 T232Q 0.33 0.57 0.89 0.83 0.98 232 T232S 0.34 0.72 0.76 0.94 1.06 232 T232V 0.50 0.67 0.89 1.42 1.42 232 T232W 0.25 0.43 0.47 0.73 0.59 232 T232Y 0.43 0.72 0.71 1.05 1.00 233 P233A 0.56 1.06 1.15 0.85 0.77 233 P233C 0.28 0.92 1.01 0.85 0.72 233 P233D 0.41 0.89 1.49 0.87 0.84 233 P233E 0.52 1.02 1.34 0.91 0.88 233 P233F 0.30 1.01 0.89 0.85 0.81 233 P233G 0.40 1.07 1.22 0.79 0.69 233 P233H 0.38 1.01 0.64 0.90 0.84 233 P233I 0.43 1.04 0.87 0.90 0.81 233 P233K 0.33 0.85 0.76 0.91 0.86 233 P233L 0.45 1.03 0.89 0.90 0.87 233 P233M 0.26 0.99 0.92 0.92 0.88 233 P233N 0.17 0.70 1.15 0.94 0.82 233 P233Q 0.35 1.03 1.19 0.94 0.87 233 P233R 0.27 0.77 0.47 0.77 0.96 233 P233S 0.32 0.60 0.62 0.98 1.01 233 P233T 0.27 0.77 0.84 0.87 0.90 233 P233V 0.33 0.76 0.80 0.78 0.92 233 P233W 0.21 0.75 0.70 0.98 0.82 233 P233Y 0.27 0.73 0.91 0.87 0.91 234 K234A 1.16 0.90 0.81 0.91 1.46 234 K234C 0.60 0.94 0.48 0.94 1.36 234 K234D 0.61 0.74 0.44 0.93 1.46 234 K234F 0.36 0.82 0.05 0.98 2.17 234 K234G 0.84 0.91 0.56 0.50 1.41 234 K234H 0.59 0.98 0.45 1.00 1.83 234 K234L 0.27 1.08 0.42 2.09 1.93 234 K234M 0.39 0.84 0.44 1.18 1.85 234 K234N 0.51 0.97 0.59 1.23 1.19 234 K234S 0.17 0.94 0.43 3.50 3.17 234 K234T 0.77 0.92 0.57 0.75 1.27 234 K234V 0.32 1.01 0.22 1.43 2.47 234 K234Y 0.28 1.01 0.39 1.29 1.86 236 A236C 0.12 0.18 0.33 3.03 3.16 236 A236D 0.39 0.97 0.91 1.03 0.61 236 A236E 0.45 1.02 0.79 0.94 0.82 236 A236F 0.42 0.96 0.62 0.95 0.93 236 A236G 0.58 0.84 0.59 0.88 0.68 236 A236H 0.62 0.97 0.54 0.93 0.90 236 A236I 0.22 0.86 0.50 0.89 0.49 236 A236K 0.34 1.08 0.56 0.95 0.69 236 A236L 0.32 0.94 0.74 0.99 0.79 236 A236M 0.41 1.01 0.71 0.88 0.90 236 A236N 0.30 0.97 0.72 0.94 0.88 236 A236P 0.14 0.29 0.22 0.38 0.87 236 A236Q 0.43 1.07 0.74 0.93 0.78 236 A236R 0.48 1.03 0.56 0.97 0.96 236 A236S 0.38 0.83 0.71 1.01 0.80 236 A236T 0.35 0.97 0.60 0.85 0.71 236 A236V 0.32 0.88 0.55 0.87 0.63 236 A236W 0.30 0.58 0.45 13.41  5.08 236 A236Y 0.42 0.54 0.53 3.39 1.31 237 N237A 0.75 1.02 0.91 0.80 0.88 237 N237C 0.43 1.26 0.77 1.02 1.28 237 N237F 0.52 0.91 0.17 0.76 1.34 237 N237G 0.71 1.06 0.38 1.02 0.95 237 N237H 0.57 0.98 0.56 0.99 1.03 237 N237I 0.66 0.96 0.23 0.68 1.05 237 N237K 0.63 1.04 0.38 1.17 1.31 237 N237L 0.76 1.04 0.58 0.64 1.40 237 N237P 0.24 0.59 0.07 1.96 3.82 237 N237Q 0.93 1.06 1.05 0.92 1.39 237 N237R 0.65 1.02 0.34 1.19 1.69 237 N237S 0.91 0.99 0.75 0.98 1.25 237 N237T 1.39 0.95 0.92 0.68 1.15 237 N237V 0.80 1.07 0.45 0.83 1.69 237 N237W 0.46 1.12 0.24 0.97 1.62 237 N237Y 0.30 0.63 0.16 2.26 3.41 239 Q239A 1.09 0.96 0.82 0.94 1.05 239 Q239C 0.63 0.99 0.94 0.98 0.97 239 Q239D 0.59 0.98 0.91 0.92 0.92 239 Q239E 0.70 1.10 1.16 1.00 1.04 239 Q239F 0.83 0.99 0.63 0.99 0.98 239 Q239G 0.39 1.21 0.58 0.91 0.97 239 Q239H 0.96 1.08 0.84 0.92 0.99 239 Q239I 0.59 1.07 0.43 0.88 0.90 239 Q239L 0.71 0.98 0.70 0.82 0.88 239 Q239M 0.85 1.08 0.77 0.97 0.99 239 Q239N 0.70 1.05 0.65 0.99 0.96 239 Q239P 0.31 0.51 0.40 0.47 0.90 239 Q239R 1.06 1.02 0.70 1.01 0.96 239 Q239S 0.88 1.08 1.04 0.90 1.02 239 Q239T 0.54 1.01 0.73 0.84 0.74 239 Q239V 0.84 0.97 0.71 1.03 0.92 239 Q239W 0.94 0.96 0.64 0.87 0.80 239 Q239Y 1.18 0.96 0.75 0.92 0.95 240 S240A 0.90 0.96 0.80 0.89 0.61 240 S240C 0.46 0.57 0.56 0.87 0.89 240 S240D 0.18

0.12 23.73  17.94  240 S240E 2.24 1.09 0.99 1.03 1.07 240 S240F 0.67 1.04 0.61 0.98 0.80 240 S240G 1.27 1.04 0.91 1.06 1.09 240 S240H 0.17 0.62 0.22 1.08 1.14 240 S240I 0.56 0.95 0.58 0.92 0.91 240 S240K 0.66 1.04 0.51 0.94 0.74 240 S240L 1.77 1.05 0.69 1.08 1.10 240 S240M 0.57 1.07 0.57 0.94 0.83 240 S240N 0.28 0.68 0.45 0.78 1.01 240 S240P 1.00 0.99 0.62 1.00 0.97 240 S240Q 0.73 0.96 0.68 0.94 0.74 240 S240R 0.66 0.68 0.48 0.98 1.67 240 S240T 0.17 0.31 0.24 0.95 1.11 240 S240V 1.16 1.08 0.73 0.95 0.97 240 S240W 0.56 0.69 0.44 1.15 1.33 240 S240Y 0.50 0.82 0.50 0.97 0.84 243 L243A 0.86 0.07 0.19 5.39 5.71 243 L243C 0.84 0.47 0.52 1.60 1.93 243 L243D 0.61 0.56 0.65 0.78 0.75 243 L243E 0.49 0.49 0.41 2.61 3.15 243 L243F 0.59 0.80 0.67 0.87 0.75 243 L243G 0.75 0.44 0.36 0.85 0.96 243 L243H

243 L243I 0.40 0.76 0.81 0.90 0.89 243 L243K 0.84 0.64 0.47 0.91 0.97 243 L243M 0.49 0.72 0.54 0.91 0.82 243 L243N 0.17 0.63 0.52 0.78 0.73 243 L243P 0.11 0.23 0.37 0.93 1.19 243 L243Q 0.58 0.75 0.67 1.04 0.95 243 L243R 0.74 0.58 0.46 1.01 0.96 243 L243S 0.95 0.58 0.44 0.86 0.74 243 L243T 0.44 0.67 0.50 0.91 0.82 243 L243V 0.34 0.36 0.42 0.75 0.80 243 L243W 0.48 0.47 0.31 0.89 0.99 243 L243Y 0.56 0.87 0.91 0.92 0.92 245 C245A 0.27 0.97 0.62 0.74 0.64 245 C245D 0.15 0.42 0.54 0.56 0.58 245 C245E 0.15 0.24 0.13 0.39 0.91 245 C245F 0.18 0.14 0.13 0.56 1.88 245 C245G 0.26 0.83 0.78 0.85 0.79 245 C245H 0.08 0.62

0.85 1.44 245 C245K 0.13 0.28 0.39 0.80 1.34 245 C245L 0.13 0.26 0.08 0.91 1.68 245 C245M 0.12 0.49 0.13 0.66 0.89 245 C245N 0.12 0.47 0.38 0.45 0.77 245 C245P 0.09 0.13 0.12 0.73 1.96 245 C245Q 0.11 0.22 0.22 0.75 1.30 245 C245R 0.14 0.20 0.35 0.40 1.61 245 C245S 0.26 0.63 0.37 0.57 0.95 245 C245T 0.13 0.46 0.28 0.74 0.88 245 C245V 0.14 0.50 0.45 0.43 0.72 245 C245W 0.15 0.14 0.20 1.16 1.54 245 C245Y 0.09 0.17 0.38 1.45 1.94 247 N247A 1.18 0.36

1.64 1.68 247 N247C 1.46 0.82 0.79 0.71 0.83 247 N247D 1.18 0.76 0.67 0.58 0.96 247 N247E 1.32 0.77 0.18 0.81 0.76 247 N247F 0.76 0.81 0.29 1.10 1.27 247 N247G 0.49 0.89 0.54 1.83 2.30 247 N247H 1.70 0.87 0.55 0.86 0.61 247 N247I 1.53 0.88 0.66 0.77 0.85 247 N247K 0.79 0.64 0.22 1.38 1.54 247 N247L 1.05 0.60 0.52 1.10 1.25 247 N247M 0.73 0.76 0.34 1.39 1.37 247 N247P 0.77 0.43

2.01 2.69 247 N247Q 0.49 0.60 0.41 1.77 1.71 247 N247R 0.71 0.46 0.16 1.64 1.85 247 N247S 0.73 0.52

2.08 1.91 247 N247T 0.62 0.88 0.68 1.16 1.39 247 N247V 0.49 0.67 0.34 2.16 1.64 247 N247Y 0.71 0.85 0.28 1.55 1.56 251 T251A 0.41 0.86 0.74 0.92 0.70 251 T251C 0.37 1.08 1.28 0.92 0.77 251 T251D 0.27 0.72 0.75 0.89 0.79 251 T251E 0.72 1.07 0.85 0.98 0.86 251 T251F 0.36 0.98 0.55 0.87 0.68 251 T251G 0.40 1.02 0.79 0.95 0.82 251 T251H 0.30 1.09 0.72 0.83 0.80 251 T251I 0.38 0.67 0.54 1.11 0.82 251 T251K 0.27 0.81 0.55 0.87 0.80 251 T251L 0.57 1.14 0.81 0.92 0.86 251 T251M 0.44 1.05 0.63 0.94 0.87 251 T251N 0.50 0.83 0.59 0.90 0.97 251 T251P 0.12 0.19

1.25 1.26 251 T251Q 0.48 1.01 0.77 0.95 0.92 251 T251R 0.58 1.02 0.62 0.96 0.89 251 T251S 0.67 1.05 0.78 0.89 0.87 251 T251V 0.91 1.04 0.77 0.99 0.98 251 T251W 0.30 0.83 0.54 0.97 0.90 251 T251Y 0.10 0.18 0.14 1.18 4.59 252 Q252A 0.43 0.95 0.85 0.95 0.73 252 Q252C 0.89 0.98 0.84 0.94 0.79 252 Q252D 0.21 0.65 0.65 1.00 0.61 252 Q252E 0.31 1.01 0.80 0.90 0.74 252 Q252F 0.39 0.82 0.52 1.15 0.72 252 Q252G 0.41 0.96 0.64 0.92 0.76 252 Q252H 0.78 0.97 0.63 0.98 0.84 252 Q252I 0.93 1.04 0.89 1.00 0.94 252 Q252K 0.60 0.63 0.53 1.53 1.18 252 Q252L 0.47 1.02 0.77 0.91 0.88 252 Q252M 0.54 1.04 0.69 0.94 0.86 252 Q252N 0.24 0.83 0.64 0.97 0.80 252 Q252P 0.16 0.12 0.09 1.25 1.12 252 Q252R 0.59 0.68 0.51 1.15 0.91 252 Q252S 0.53 1.01 0.82 0.92 0.88 252 Q252T 0.32 0.69 0.33 0.55 0.62 252 Q252V 0.52 0.83 0.40 1.12 1.12 252 Q252W 0.34 0.66 0.60 1.18 0.85 252 Q252Y 0.51 0.98 0.72 0.95 0.85 254 N254A 0.28 0.88 0.69 0.91 0.88 254 N254C 0.26 0.88 0.82 0.83 0.91 254 N254E 0.20 0.84 0.56 0.76 0.91 254 N254F 0.15 0.33 0.10 0.72 1.71 254 N254G 0.28 0.37 0.29 0.73 1.31 254 N254I 0.36 0.18 0.15 0.57 1.14 254 N254K 0.24 0.24 0.44 0.45 1.18 254 N254L 0.23 0.30 0.11 0.49 0.64 254 N254M 0.18 0.33 0.33 0.89 1.34 254 N254P 0.36 0.22 0.05 0.98 1.35 254 N254Q 0.39 0.26 0.14 0.85 1.07 254 N254R 0.36 0.14 0.08 1.49 1.94 254 N254S 0.29 0.49 0.19 0.87 1.24 254 N254T 0.22 0.53 0.50 0.97 1.34 254 N254V 1.22 0.98 0.90 1.05 1.08 254 N254W 0.26 0.17 0.12 1.05 1.02 254 N254Y 0.21 0.40 0.05 0.88 1.30 258 V258A 0.56 0.63 0.72 1.10 1.08 258 V258C 0.52 0.74 0.68 1.09 1.41 258 V258D 0.20 0.28 0.55 1.07 1.24 258 V258E 0.18 0.17 0.29 1.03 1.45 258 V258F 0.12 0.19 0.24 0.87 2.94 258 V258G 0.18 0.50 0.48 0.83 0.88 258 V258H 0.16 0.20 0.43 1.16 1.55 258 V258I 0.26 0.87 0.79 0.85 0.78 258 V258K 0.17 0.32 0.42 0.49 0.89 258 V258L 0.34 0.92 1.10 0.88 0.82 258 V258M 0.33 0.91 1.11 0.87 0.86 258 V258N 0.22 0.47 0.46 0.95 0.88 258 V258P 0.21 0.13 0.19 1.53 1.46 258 V258Q 0.18 0.23 0.38 0.53 1.08 258 V258R 0.23 0.15

0.33 1.69 258 V258S 0.34 0.73 0.83 0.90 0.89 258 V258T 0.64 0.98 1.01 0.94 0.91 258 V258W 0.21 0.16 0.37 0.88 1.85 258 V258Y 0.18 0.14 0.14 1.13 1.36 266 H266A 0.26 0.41 0.39 0.42 0.57 266 H266C 0.08 0.15 0.28 1.20 2.86 266 H266D 0.17 0.24 0.23 1.06 1.47 266 H266E 0.20 0.20 0.30 2.06 3.59 266 H266I 0.21 0.14 0.16 3.49 1.64 266 H266K 0.18 0.25 0.13 1.55 0.94 266 H266L 0.24 0.14 0.20 0.85 1.74 266 H266M 0.18 0.19 0.29 6.85 0.91 266 H266N 0.49 0.73 0.48 0.79 0.63 266 H266P 0.14 0.14 0.26 0.98 1.25 266 H266R 0.30 0.17 0.23 1.16 1.30 266 H266S 0.19 0.29 0.26 0.68 0.87 266 H266T 0.20 0.27 0.18 2.35 1.95 266 H266V 0.21 0.20 0.30 0.67 1.54 266 H266W 0.22 0.27 0.11 0.86 0.32 266 H266Y 0.24 0.30 0.22 0.50 0.54 267 A267C 0.85 0.97 0.75 0.99 0.83 267 A267D 0.23 0.23 0.07 0.76 0.91 267 A267E 0.34 0.52 0.44 0.56 0.41 267 A267F 0.21 0.12

1.58 0.93 267 A267G 0.86 1.00 0.36 0.44 0.13 267 A267H 0.38 0.35

0.52 0.34 267 A267I 0.21 0.11

0.82 0.86 267 A267K 0.20 0.21

1.20 1.07 267 A267L 0.21 0.19 0.08 0.84 1.56 267 A267M 0.23 0.22 0.06 0.71 0.73 267 A267N 0.29 0.49

0.47 0.45 267 A267P 0.21 0.28 0.14 0.72 0.95 267 A267R 0.18 0.16 0.20 1.08 0.95 267 A267S 0.80 0.93 0.67 0.83 0.55 267 A267T 0.34 0.68 0.17 0.46 0.24 267 A267V 0.23 0.52 0.21 0.28 0.31 267 A267W 0.25 0.22 0.16 0.91 0.83 267 A267Y 0.21 0.19 0.05 0.80 0.85 268 G268A 4.40 0.33 0.14 1.65 1.34 268 G268C 0.53 0.49 0.19 1.23 1.99 268 G268D 0.61 0.74 0.80 0.73 0.55 268 G268E 0.55 0.17 0.07 1.42 1.95 268 G268F 1.01 0.47 0.41 0.94 1.00 268 G268H 1.25

0.35

268 G268I 0.76 0.65 0.80 1.21 0.84 268 G268K 0.86 0.75 0.77 0.83 0.75 268 G268L 0.76 0.44 0.27 1.10 1.31 268 G268M 0.55 0.36 0.14 0.49 0.31 268 G268N 0.62 0.37 0.33 0.86 1.32 268 G268P 0.49 0.53 0.53 1.16 1.29 268 G268Q 1.37 0.53 0.42 0.85 0.94 268 G268R 1.09 0.40 0.37 0.95 0.82 268 G268S 0.81 0.70 0.36 0.53 0.52 268 G268T 0.69 0.42 0.29 0.76 0.74 268 G268V 1.23 0.47 0.33 0.97 0.98 268 G268W 0.69 0.47 0.47 1.71 2.59 268 G268Y 0.61 0.25 0.06 0.76 1.28 272 W272A 0.93 0.52 0.20 0.59 0.33 272 W272C 0.57 0.43 0.13 0.40 0.34 272 W272D 0.84 0.51 0.24 0.41 0.10 272 W272E 0.67 0.47 0.12 0.55 0.41 272 W272F 0.59 0.73 0.23 0.72 0.46 272 W272G 0.89 0.54 0.13 0.74 0.43 272 W272H 0.07 0.27

1.91 0.37 272 W272I 0.67 0.55 0.16 0.44 0.20 272 W272K 0.74 0.31

0.35 0.53 272 W272L 0.67 0.59 0.20 0.53 0.42 272 W272M 1.42 0.62 0.14 0.61 0.32 272 W272N 0.92 0.51 0.10 0.48 0.33 272 W272P 0.61 0.49 0.14 0.62 0.44 272 W272Q 0.14 0.07 0.11 0.63 0.92 272 W272R 0.89 0.36

0.58 0.44 272 W272S 1.03 0.51 0.32 0.75 0.43 272 W272T 0.85 0.49 0.18 0.52 0.31 272 W272V 0.74 0.53 0.07 0.27 0.15 272 W272Y 0.85 0.67 0.28 0.50 0.45 274 A274C 0.23 0.47 0.52 2.95 0.43 274 A274D 0.16 0.58 0.55 0.79 0.43 274 A274E 0.32 0.99 0.87 0.98 0.80 274 A274F 0.33 0.85 0.42 0.91 0.89 274 A274G 0.34 1.02 0.53 0.82 0.65 274 A274H 0.40 0.83 0.37 0.91 0.67 274 A274I 0.26 0.88 0.34 0.84 0.71 274 A274K 0.26 0.78 0.33 0.91 0.85 274 A274L 0.42 0.88 0.49 0.96 0.75 274 A274M 0.31 0.63 0.48 1.23 0.84 274 A274N 0.41 0.97 0.67 0.95 0.89 274 A274P 0.26 0.74 0.43 0.95 0.51 274 A274Q 0.44 0.86 0.73 0.97 0.98 274 A274R 0.51 0.99 0.38 0.97 0.91 274 A274S 0.46 0.96 0.71 0.97 0.84 274 A274T 0.41 0.55 0.47 1.14 1.03 274 A274V 0.38 0.87 0.51 0.96 0.85 274 A274W 0.56 0.70 0.37 1.03 0.88 274 A274Y 0.11 0.07 0.10 1.25 0.33 275 N275A 1.11 0.93 0.87 1.02 0.97 275 N275C 0.74 0.76 0.71 1.01 0.95 275 N275D 0.40 0.78 0.68 0.97 1.00 275 N275E 0.65 0.90 0.57 1.02 0.96 275 N275F 1.34 0.86 0.71 0.98 0.98 275 N275G 0.20 0.67 0.17 0.31 0.18 275 N275K 0.27 0.84 0.34 0.91 0.94 275 N275L 0.98 0.90 0.70 0.92 1.00 275 N275M 0.65 0.87 0.63 1.04 0.98 275 N275Q 0.58 0.88 0.56 1.03 0.95 275 N275R 0.64 0.68 0.27 0.96 1.04 275 N275S 0.61 0.99 0.57 0.91 0.87 275 N275T 0.70 0.85 0.53 0.96 1.01 275 N275V 0.81 0.89 0.53 0.95 0.97 275 N275W 1.07 0.92 0.69 1.00 1.02 275 N275Y 0.37 0.64 0.43 0.94 0.96 281 Q281A 1.53 0.81 0.66 0.85 0.32 281 Q281C 1.70 0.88 0.70 0.73 0.43 281 Q281D 1.10 0.60 0.43 1.33 0.95 281 Q281E 2.50 0.89 0.85 1.10 0.63 281 Q281F 0.76 0.72 0.15 1.31 0.97 281 Q281G 1.04 0.75 0.46 1.02 1.35 281 Q281I 0.62 0.63 0.08 1.74 1.66 281 Q281L 0.78 0.68 0.31 1.79 1.51 281 Q281N 1.38 0.85 0.46 1.09 0.85 281 Q281T 0.59 0.72 0.22 1.64 1.10 281 Q281V 1.46 0.94 0.69 1.22 0.86 285 N285A 0.88 1.01 0.71 0.50 0.62 285 N285C 0.46 1.02 0.70 1.22 1.23 285 N285D 0.35 0.70 0.59 1.99 2.24 285 N285F 0.27 0.67 0.15 2.16 1.85 285 N285G 0.47 1.24 0.62 1.02 0.92 285 N285H 0.60 1.04 0.30 0.93 1.11 285 N285K 0.36 0.76 0.25 1.49 2.79 285 N285L 0.46 1.15 0.68 1.16 1.05 285 N285P 0.18 0.27 0.18 6.33 7.75 285 N285S 0.26 0.77 0.21 2.14 2.16 285 N285T 0.40 0.78 0.33 1.66 2.14 285 N285V 0.19 0.84 0.18 2.84 3.86 285 N285W 0.39 1.21 0.38 1.60 2.07 285 N285Y 0.72 1.15 1.19 1.15 0.91 288 K288A 0.41 0.61 0.68 1.52 2.84 288 K288C 0.41 0.95 0.63 0.72 1.49 288 K288D 0.23 0.23

5.77 5.10 288 K288E 0.38 0.89 0.81 0.65 1.60 288 K288F 0.36 0.75 0.29 1.53 2.74 288 K288G 0.23 0.40 0.12 2.22 4.09 288 K288H 0.32 0.90 0.48 1.09 1.54 288 K288I 0.40 0.75 0.37 1.51 2.48 288 K288L 0.80 0.96 0.81 0.60 0.82 288 K288N 0.71 0.98 0.84 1.08 1.68 288 K288P 0.26 0.34

5.00 5.39 288 K288Q 0.23 0.80 0.18 1.23 2.51 288 K288R 0.27 0.42

2.13 3.33 288 K288S 0.19 0.71 0.21 2.02 2.72 288 K288T 0.25 0.71 0.35 1.27 2.96 288 K288V 0.31 0.84 0.38 1.67 1.87 289 N289A 0.39 0.70 0.90 0.98 0.87 289 N289C 0.21 0.10 0.50 6.35 7.25 289 N289D 0.84 0.96 0.87 0.93 0.92 289 N289E 0.13 0.26 0.63 1.03 1.48 289 N289F 0.14 0.27 0.39 0.85 0.78 289 N289G 0.17 0.42 0.79 0.72 0.58 289 N289H 0.15 0.54 0.66 0.94 0.91 289 N289I 0.16 0.20

0.64 0.68 289 N289K 0.31 0.79 0.66 0.88 0.85 289 N289L 0.16 0.39 0.48 0.92 1.00 289 N289M 0.18 0.52 0.14 0.80 0.74 289 N289Q 0.18 0.49 0.45 0.63 0.66 289 N289R 0.41 0.87 0.23 0.91 0.84 289 N289S 0.44 1.05 0.85 0.84 0.88 289 N289T 0.20 0.58 0.79 0.90 0.86 289 N289V 0.22 0.64 0.33 0.92 0.83 289 N289W 0.19 0.43 0.59 0.95 0.98 289 N289Y 0.24 0.62 0.47 0.78 0.91 291 S291A 1.22 2.27 2.57 0.90 1.26 291 S291C 2.02 2.57 3.26 0.99 1.96 291 S291D 0.40 0.28 0.24 1.20 5.19 291 S291E 2.53 2.54 3.48 1.02 1.91 291 S291F 1.57 2.22 1.63 1.02 1.89 291 S291G 0.60 0.24 0.25 1.08 0.92 291 S291H 2.52 2.45 2.33 1.06 1.94 291 S291I 0.81 0.95 0.53 1.09 1.32 291 S291K 1.26 2.23 1.10 1.05 1.58 291 S291L 1.28 1.99 1.50 0.91 1.23 291 S291M 1.88 2.50 2.13 1.01 1.91 291 S291N 0.46 0.21

2.48 2.00 291 S291P 0.75 0.72 0.88 1.18 1.19 291 S291T 1.56 2.62 2.22 1.11 2.02 291 S291V 0.99 0.61 0.24 1.25 1.24 291 S291W 1.09 2.20 1.87 1.01 1.50 291 S291Y 0.90 1.18 0.85 0.95 1.48 292 S292A 0.96 0.97 1.12 0.99 0.94 292 S292C 0.44 0.53 0.61 1.35 1.67 292 S292D 0.65 0.79 0.74 0.91 1.11 292 S292E 0.50 0.73 0.47 1.14 1.40 292 S292F 0.74 0.87 0.48 0.86 1.05 292 S292G 0.67 0.99 0.68 0.74 0.80 292 S292H 0.35 0.67 0.63 1.16 1.90 292 S292I 0.41 0.79 0.46 0.62 0.83 292 S292K 0.85 0.84 0.72 0.78 0.75 292 S292L 0.31 0.61 0.65 0.59 0.60 292 S292M 0.75 0.99 0.74 0.72 0.87 292 S292N 0.69 0.99 0.75 0.90 1.06 292 S292P 0.42 0.52 0.34 0.53 0.85 292 S292Q 0.55 0.87 0.58 0.97 0.79 292 S292R 1.12 0.98 0.45 0.84 0.85 292 S292T 0.84 1.01 0.65 0.86 0.92 292 S292V 0.44 0.70 0.51 0.95 1.25 292 S292W 0.91 1.10 0.82 0.83 0.64 292 S292Y 1.20 0.94 0.62 1.00 0.91 293 P293A 0.34 0.93 0.85 0.94 0.76 293 P293C 0.21 0.46 0.51 0.96 0.58 293 P293D 0.30 0.14 0.25 0.25 0.54 293 P293E 0.13 0.14 0.23 0.94 1.08 293 P293F 0.16 0.18 0.22 0.64 0.94 293 P293G 0.19 0.40 0.59 0.72 0.53 293 P293H 0.17 0.14 0.14 0.62 1.01 293 P293I 0.17 0.29 0.30 0.65 0.92 293 P293K 0.13 0.21 0.53 4.29 0.89 293 P293L 0.16 0.27 0.21 0.80 0.51 293 P293M 0.15 0.24 0.12 5.43 0.67 293 P293N 0.16 0.31 0.24 0.63 0.92 293 P293Q 0.22 0.16 0.17 0.29 0.54 293 P293R 0.15 0.31 0.62 1.31 0.86 293 P293S 0.18 0.38 0.24 1.04 0.74 293 P293T 0.18 0.28 0.12 0.44 0.78 293 P293V 0.17 0.77 0.60 0.93 0.73 293 P293W 0.18 0.11 0.16 2.77 9.61 293 P293Y 0.13 0.08 0.11 0.31 1.24 294 R294A 0.97 1.06 0.89 0.92 0.77 294 R294C 0.70 1.15 0.74 1.01 1.16 294 R294D 0.75 0.94 0.85 0.94 1.09 294 R294E 0.73 0.82 0.66 1.02 1.20 294 R294F 0.37 0.52 0.54 0.90 1.56 294 R294G 1.25 0.93 0.84 0.71 0.87 294 R294H 0.96 0.93 0.55 0.97 1.18 294 R294I 0.77 0.84 0.72 0.98 1.01 294 R294K 1.34 0.94 0.69 1.04 1.20 294 R294L 0.54 1.06 1.03 1.05 1.22 294 R294M 0.34 0.76 0.78 0.81 1.78 294 R294N 0.50 0.80 0.64 1.30 1.06 294 R294P 0.48 0.86 0.74 0.74 0.99 294 R294Q 0.50 0.79 0.75 1.23 1.30 294 R294S 0.64 1.00 0.57 1.32 1.47 294 R294T 1.12 1.03 0.97 1.02 1.10 294 R294V 0.83 0.96 0.83 0.82 1.03 294 R294W 1.08 1.01 0.57 0.56 0.58 294 R294Y 0.26 0.43 0.31 2.39 3.19 303 V303A 0.30 0.46 0.33 0.40 0.38 303 V303E 0.21 0.12 0.24 2.70 4.92 303 V303F 0.22 0.14 0.16 0.70 0.93 303 V303G 0.23 0.24 0.29 0.48 0.86 303 V303H 0.29 0.13 0.31 0.84 2.17 303 V303I 0.85 1.07 0.68 0.67 0.37 303 V303K 0.24 0.15 0.31 1.05 1.94 303 V303L 0.29 0.69 0.19 0.17 0.16 303 V303M 0.18 0.29 0.13 0.47 44.68  303 V303N 0.25 0.30 0.25 0.46 0.65 303 V303P 0.30 0.13 0.13 1.31 2.70 303 V303Q 0.32 0.16 0.20 1.08 1.32 303 V303R 0.19 0.14 0.21 1.74 1.78 303 V303S 0.26 0.37 0.14 1.00 0.83 303 V303T 0.39 0.98 0.48 0.70 0.45 303 V303W 0.18 0.15 0.23 1.50 1.97 303 V303Y 0.23 0.19 0.15 2.71 1.26 304 A304C 0.76 0.47 0.36 0.62 0.39 304 A304D 1.25 0.13 0.16 15.46  16.85  304 A304E 0.32 0.08 0.30 3.56 3.07 304 A304F 0.49 0.08 0.15 1.43 1.58 304 A304G 1.16 0.65 0.42 0.80 0.66 304 A304H 0.73 0.09 0.28 0.97 1.38 304 A304I 0.66 0.15 0.39 1.93 1.79 304 A304K 0.82 0.15 0.17 1.63 1.59 304 A304L 0.52 0.10 0.25 1.55 2.80 304 A304M 0.82 0.11 0.12 1.89 1.39 304 A304N 0.65 0.11 0.14 5.49 17.77  304 A304P 0.31 0.11 0.14 1.16 2.23 304 A304Q 0.66 0.13 0.22 2.05 1.82 304 A304R 1.17 0.14 0.22 1.45 1.27 304 A304S 0.42 0.83 0.82 0.83 0.67 304 A304T 0.58 0.19 0.22 1.08 1.33 304 A304V 0.53 0.20 0.31 0.80 1.47 304 A304W 0.50 0.12 0.23 1.41 2.07 304 A304Y 0.54

0.32 3.22 4.73 306 Y306A 0.35 0.77 0.39 0.10 0.15 306 Y306C 0.16 0.49 0.27 0.21 0.91 306 Y306D 0.18 0.39 0.31 0.15 0.30 306 Y306E 0.15 0.45 0.22 0.15 0.46 306 Y306G 0.16 0.45 0.18 0.32 0.59 306 Y306H 0.32 0.60 0.19 0.07 0.39 306 Y306I 0.34 0.81 0.23 0.09 0.17 306 Y306K 0.19 0.17 0.28 0.41 0.58 306 Y306L 0.16 0.55 0.29 0.16 0.47 306 Y306N 0.22 0.60 0.13 0.08 0.21 306 Y306P 0.21 0.28 0.22 0.34 0.74 306 Y306Q 0.23 0.70 0.17 0.11 0.25 306 Y306R 0.20 0.51 0.23 0.18 0.35 306 Y306S 0.35 0.80 0.26 0.06 0.15 306 Y306T 0.60 0.91 0.17 0.09 0.13 306 Y306V 0.35 0.82 0.17 0.09 0.12 306 Y306W 0.50 0.78 0.68 0.56 0.75 307 N307A 0.13 0.23 0.25 0.54 0.73 307 N307C 0.17 0.34 0.22 0.19 0.38 307 N307D 0.20 0.77 0.57 0.15 0.24 307 N307E 0.11 0.56 0.16 0.53 0.82 307 N307F 0.13 0.24 0.20 0.43 0.88 307 N307G 0.25 0.59 0.25 0.20 0.52 307 N307H 0.15 0.22 0.23 0.40 0.61 307 N307I 0.23 0.42 0.20 0.15 0.33 307 N307K 0.14 0.21 0.26 0.38 1.07 307 N307L 0.12 0.30 0.21 0.34 0.69 307 N307M 0.15 0.33 0.17 0.33 0.77 307 N307P 0.14 0.15 0.23 0.83 2.18 307 N307Q 0.17 0.44 0.25 0.19 0.59 307 N307R 0.18 0.21 0.17 0.56 0.85 307 N307S 0.27 0.70 0.24 0.14 0.25 307 N307T 0.14 0.31 0.12 0.53 0.74 307 N307V 0.15 0.29 0.25 0.27 0.82 307 N307W 0.20 0.21 0.11 0.61 0.99 307 N307Y 0.20 0.18 0.19 0.30 1.06 312 T312A 1.35 2.81 1.85 0.20 0.23 312 T312C 0.80 0.73 0.48 0.67 0.88 312 T312D 1.09 1.51 0.93 0.22 0.28 312 T312E 1.39 2.50 1.93 0.31 0.13 312 T312F 1.07 1.90 0.98 0.25 0.05 312 T312G 1.17 2.33 0.47 0.16 0.27 312 T312H 0.98 1.48 0.64 0.70 0.46 312 T312I 1.21 2.10 0.21 0.20 0.06 312 T312K 0.78 2.31 0.87 0.36 0.24 312 T312L 0.76 0.69 0.23 1.03

312 T312N 0.66 0.82 0.30 0.80 0.22 312 T312P 1.18 2.16 0.42 0.29 0.14 312 T312Q 0.81 1.81 0.48 0.43 0.25 312 T312R 1.20 2.32 0.76 0.32 0.32 312 T312S 1.32 2.02 1.79 0.92 1.28 312 T312V 0.35 0.31 0.10 1.17 2.92 312 T312W 1.11 1.34 0.32 0.19 0.38 312 T312Y 1.05 1.62 0.71 0.24 0.13 313 S313A 0.34 1.08 0.85 0.77 0.60 313 S313C 0.34 0.94 0.58 0.79 0.84 313 S313D 0.43 1.04 1.32 1.01 0.99 313 S313E 0.46 1.20 0.90 0.99 0.88 313 S313F 0.59 1.02 0.54 0.82 0.78 313 S313G 0.73 1.05 0.30 0.30 0.13 313 S313I 0.69 1.06 0.69 0.90 0.90 313 S313K 1.64 1.07 0.53 1.04 1.16 313 S313L 0.86 1.08 0.55 0.62 0.29 313 S313M 0.50 1.12 0.72 0.74 0.57 313 S313P 1.00 0.95 0.55 0.56 0.31 313 S313Q 0.74 1.04 0.81 0.93 1.01 313 S313R 0.55 1.05 0.37 0.83 0.76 313 S313T 0.98 1.15 1.21 1.00 1.16 313 S313V 0.53 1.05 0.81 0.87 0.82 313 S313W 0.70 1.00 0.63 0.78 0.46 313 S313Y 0.52 0.97 0.49 0.67 0.31 316 S316A 0.59 0.96 1.36 1.49 1.15 316 S316C 0.77 0.87 1.10 1.43 1.15 316 S316D 0.85 0.98 1.18 0.63 0.46 316 S316E 0.13 0.12

1.22 1.85 316 S316F 0.65 0.99 0.68 1.06 0.83 316 S316G 0.69 0.97 0.60 0.71 0.79 316 S316H 0.63 1.05 0.48 1.28 0.80 316 S316I 0.50 0.90 0.51 1.62 0.91 316 S316K 0.78 0.99 0.66 0.97 0.85 316 S316L 1.27 1.00 1.19 0.99 0.88 316 S316M 0.15 0.07 0.15 14.10  22.76  316 S316N 0.73 1.06 0.92 0.76 0.70 316 S316P 0.98 0.95 1.46 1.74 1.48 316 S316Q 0.82 0.97 0.91 0.85 0.74 316 S316R 1.18 0.93 0.78 1.20 0.76 316 S316T 0.92 0.92 0.89 0.89 0.68 316 S316V 0.76 0.98 0.88 1.12 0.94 316 S316W 1.13 0.92 1.16 1.54 1.39 316 S316Y 0.85 0.93 0.60 0.56 0.87 319 Q319A 0.55 1.03 0.96 1.06 1.00 319 Q319C 0.52 0.95 0.76 1.04 0.91 319 Q319D 0.78 1.01 0.86 0.95 0.80 319 Q319E 0.83 1.08 0.92 1.03 0.88 319 Q319F 0.71 0.98 0.40 1.06 0.94 319 Q319G 0.81 0.98 0.41 0.78 0.56 319 Q319I 0.71 1.04 0.60 1.03 1.01 319 Q319K 0.84 1.00 0.45 1.04 1.12 319 Q319L 0.48 1.03 0.61 1.10 1.13 319 Q319M 0.52 1.03 0.89 0.96 0.98 319 Q319N 0.81 1.05 0.70 0.96 0.92 319 Q319P 0.44 0.91

0.26 0.30 319 Q319R 0.71 1.01 0.49 1.06 1.14 319 Q319S 1.06 1.03 0.69 0.99 1.00 319 Q319T 0.93 1.06 0.77 0.97 0.95 319 Q319V 0.65 1.02 0.69 1.00 1.02 319 Q319W 0.50 0.85 0.59 1.06 0.99 319 Q319Y 1.02 1.01 0.81 1.01 1.05 322 A322C 0.52 0.98 0.98 0.95 0.85 322 A322E 0.56 1.02 1.05 0.99 0.98 322 A322F 0.58 0.99 0.64 0.81 0.69 322 A322G 0.68 0.94 0.70 0.97 0.78 322 A322H 0.48 0.97 0.50 0.91 0.84 322 A322I 0.58 0.98 0.79 0.96 0.94 322 A322K 0.56 1.00 0.56 0.95 1.02 322 A322L 0.57 1.00 0.57 0.94 0.93 322 A322M 0.81 1.05 0.97 1.00 0.96 322 A322N 0.45 0.98 0.73 0.92 0.96 322 A322P 0.88 1.02 1.06 1.09 1.21 322 A322Q

322 A322R 0.51 1.01 0.33 1.01 1.01 322 A322S 0.53 1.00 1.00 1.03 1.01 322 A322T 0.91 1.03 0.91 0.99 1.01 322 A322V 0.62 1.02 0.93 0.93 1.00 322 A322W 0.47 0.91 0.44 0.87 0.76 322 A322Y 0.45 0.93 0.56 0.90 0.82 323 V323A 0.89 1.01 0.80 0.61 0.62 323 V323C 0.73 1.05 1.21 1.51 0.84 323 V323D 0.48 0.79 0.46 0.88 0.81 323 V323E 0.49 0.76 0.59 0.80 0.79 323 V323F 0.68 0.92 0.86 1.09 0.85 323 V323G 0.30 0.84 0.27 1.23 1.27 323 V323H 0.67 0.90 0.27 0.66 0.56 323 V323I 0.70 1.07 1.04 0.53 0.73 323 V323K 0.38 0.93 0.51 1.34 1.11 323 V323L 0.57 0.99 0.22 0.60 0.68 323 V323N 0.47 1.01 0.38 0.88 1.04 323 V323Q 0.42 0.97 0.47 0.72 1.05 323 V323R 0.23 0.69 0.26 2.20 2.25 323 V323S 0.30 0.80 0.09 1.00 1.42 323 V323T 0.60 0.80

0.66 1.05 323 V323W 0.45 0.90 0.20 0.71 1.06 323 V323Y 0.35 1.00 0.39 0.82 1.16 327 K327A 0.86 1.03 0.98 0.76 0.76 327 K327C 0.42 0.93 1.25 1.28 1.02 327 K327D 0.31 0.73 0.76 1.13 1.81 327 K327E 0.73 0.84 0.85 1.05 0.87 327 K327F 0.24 0.61 0.44 1.91 2.14 327 K327G 0.24 0.83 0.16 1.65 1.30 327 K327H 0.58 1.05 1.22 0.66 0.80 327 K327I 0.31 0.74 0.23 1.44 1.37 327 K327L 0.26 0.94 0.60 0.89 1.30 327 K327M 0.41 0.90 0.90 0.87 1.44 327 K327N 0.41 0.90 1.04 0.74 1.33 327 K327P 0.21 0.58

1.65 2.77 327 K327Q 1.32 1.06 1.42 1.15 0.77 327 K327R 0.56 0.85 0.89 1.07 1.16 327 K327S 0.20 0.63 0.58 2.39 2.92 327 K327T 0.21 0.29 0.09 3.69 7.57 327 K327V 0.28 0.86 0.58 0.66 1.26 327 K327W 0.28 1.05 0.74 0.93 1.57 327 K327Y 0.93 1.04 1.08 0.49 0.60 328 L328A 0.91 1.11 0.89 0.49 0.18 328 L328C

328 L328D 0.95 1.01 0.55 0.21 0.06 328 L328E 0.97 0.97 0.54 0.31 0.10 328 L328F 0.53 0.92 0.27 0.47 0.21 328 L328G 0.68 0.96 0.55 0.61 0.30 328 L328H 0.62 1.05 0.27 0.22 0.16 328 L328I 0.67 1.01 0.72 0.82 0.58 328 L328K 0.91 1.02 0.45 0.88 0.77 328 L328M 0.91 0.95 0.61 0.89 0.82 328 L328N 0.86 0.90 0.44 0.49 0.22 328 L328P 0.25 0.58 0.17 0.24 0.29 328 L328Q 2.73 1.07 0.86 0.85 0.76 328 L328R 0.74 0.92 0.34 0.81 0.62 328 L328S 0.40 0.90 0.42 0.38 0.18 328 L328T 0.57 0.93 0.50 0.30 0.13 328 L328V 0.40 0.74 0.20 0.14 0.10 328 L328W 0.45 0.88 0.27 0.12 0.14 328 L328Y

331 H331A 0.31 0.58 0.60 0.52 0.18 331 H331C 0.36 0.17 0.32 0.70 0.09 331 H331D 0.46 0.79 0.66 0.50 0.15 331 H331E 0.34 0.66 0.60 0.64 0.36 331 H331F 0.29 0.23 0.13 0.33

331 H331G 0.29 0.68 0.51 0.57 0.24 331 H331I 0.25 0.14 0.26 0.06 12.66  331 H331K 0.39 0.79 0.60 0.50 0.14 331 H331L 0.24 0.36 0.31 0.37 0.20 331 H331M 0.25 0.41 0.29 0.18 0.07 331 H331N 0.58 0.98 1.06 0.82 0.64 331 H331P 0.25 0.09 0.30 0.26 1.41 331 H331Q 0.40 0.84 0.92 0.62 0.35 331 H331R 0.38 0.67 0.43 0.53 0.21 331 H331S 0.33 0.56 0.55 0.74 0.35 331 H331T 0.24 0.64 0.58 0.57 0.27 331 H331V 0.22 0.25 0.30 0.16 0.22 331 H331W 0.32 0.69 0.69 0.66 0.44 331 H331Y 0.58 0.81 0.72 0.68 0.36 338 A338C 0.79 0.94 0.81 0.93 0.91 338 A338D 1.11 0.97 0.90 1.03 1.06 338 A338E 0.71 0.97 0.74 0.95 0.93 338 A338F 0.56 0.90 0.72 0.92 0.88 338 A338G 0.71 0.98 0.75 0.93 0.92 338 A338H 0.85 1.05 0.57 0.94 1.02 338 A338I 0.74 0.97 0.87 1.00 1.14 338 A338K 0.90 1.05 0.67 0.98 1.02 338 A338L 0.36 0.68 0.71 1.02 0.97 338 A338M 0.72 0.88 0.84 1.10 1.15 338 A338N 0.87 1.02 0.81 0.97 1.04 338 A338P 0.48 0.88 0.85 0.97 1.03 338 A338Q 0.34 0.66 0.57 0.92 0.86 338 A338R 0.70 1.09 0.79 0.95 0.97 338 A338S 0.70 0.94 0.86 1.00 1.05 338 A338T 0.21 0.68 0.68 0.83 0.88 338 A338V 0.48 1.00 1.01 0.92 1.01 338 A338W 0.30 0.83 0.75 0.87 0.94 339 N339A 0.67 1.03 1.24 1.06 1.10 339 N339C 0.60 0.98 1.09 0.96 0.96 339 N339D 0.92 1.04 1.05 1.04 1.10 339 N339E 0.94 1.03 0.83 1.08 1.13 339 N339F 0.53 1.02 0.56 1.08 1.12 339 N339G 0.57 1.01 0.63 1.09 1.00 339 N339H 0.66 1.05 0.58 1.00 1.11 339 N339I 0.66 1.09 0.77 1.05 1.13 339 N339K 0.69 1.03 0.65 1.06 1.11 339 N339L 0.61 1.13 0.74 1.05 1.15 339 N339M 0.47 1.03 0.51 1.05 1.09 339 N339P 0.19 0.90 0.64 0.84 0.74 339 N339Q 0.36 1.06 0.69 1.06 0.84 339 N339R 0.97 1.01 0.58 1.07 1.10 339 N339S 0.74 1.04 0.85 0.97 1.02 339 N339T 0.71 1.03 0.91 0.98 1.00 339 N339V 0.75 1.08 0.65 1.04 1.08 339 N339W 0.17 0.46 0.44 0.83 1.26 339 N339Y 0.59 0.97 0.73 0.88 0.81 340 H340A 0.25 0.18 0.44 0.23

340 H340C 0.39 0.69 0.72 0.65 0.37 340 H340D 0.32 0.45 0.78 0.41 0.28 340 H340E 0.39 0.73 0.79 0.80 0.51 340 H340F 0.68 1.08 0.91 0.77 0.64 340 H340G 0.35 0.44 0.50 0.47 0.24 340 H340I 0.28 0.29 0.44 0.51 0.14 340 H340K 0.71 1.03 1.00 0.83 0.69 340 H340L 0.36 0.84 0.70 0.63 0.42 340 H340M 0.36 0.73 0.68 0.81 0.52 340 H340N 0.76 0.92 1.02 0.92 0.81 340 H340P 0.22 0.17 0.21 0.16 0.39 340 H340Q 0.33 0.69 0.49 0.66 0.42 340 H340R 0.22 0.12 0.36 0.13 0.13 340 H340S 0.36 0.81 0.83 0.65 0.46 340 H340T 0.24 0.36 0.31 0.28 0.14 340 H340V 0.31 0.48 0.60 0.52 0.26 340 H340W 1.02 1.03 0.97 0.88 0.80 340 H340Y 0.38 0.85 1.01 0.83 0.66 343 S343A 1.40 2.48 2.75 0.94 1.77 343 S343C 3.71 2.52 3.51 1.02 2.16 343 S343D 1.37 2.87 3.53 0.95 1.67 343 S343E 4.68 2.37 3.30 1.09 2.31 343 S343F 1.52 2.15 1.82 0.95 1.59 343 S343G 0.51 0.48 0.19 3.20 5.39 343 S343H 0.66 0.73 0.33 0.97 2.31 343 S343I 1.19 2.45 1.98 1.09 1.76 343 S343K 2.34 2.56 3.03 1.19 2.04 343 S343L 1.32 2.65 2.59 1.00 1.83 343 S343M 0.18 0.57 0.73 6.24 57.92  343 S343P 2.09 2.50 2.38 1.01 2.03 343 S343Q 3.31 2.39 2.38 1.12 2.29 343 S343R 1.02 2.73 1.69 1.22 1.75 343 S343T 4.19 2.37 3.08 1.15 2.26 343 S343V 0.47 0.49 0.17 1.38 2.43 343 S343W 2.83 2.47 2.53 1.12 2.19 343 S343Y 2.20 2.50 2.89 1.07 2.01 344 N344A 0.58 1.02 1.17 1.13 1.12 344 N344C 0.46 0.96 1.31 0.84 0.96 344 N344D 0.29 0.85 1.10 1.43 1.31 344 N344F 0.31 0.87 0.42 1.03 1.08 344 N344G 0.73 1.02 1.25 1.17 1.25 344 N344H 0.64 0.96 0.36 0.81 0.90 344 N344I 0.26 0.78 0.60 1.33 1.49 344 N344K 0.89 1.04 0.91 0.90 0.72 344 N344L 0.23 0.69 0.40 1.49 2.90 344 N344M 0.53 0.99 0.79 0.60 0.75 344 N344P 0.33 0.87 0.61 1.62 1.82 344 N344Q 0.69 1.01 0.68 1.07 1.06 344 N344R 0.28 0.87 0.30 1.45 1.62 344 N344S 0.84 0.94 0.77 0.98 0.92 344 N344T 0.27 0.67 0.39 1.51 2.53 344 N344V 0.26 0.82 0.42 1.44 1.84 344 N344W 0.22 0.64

1.86 2.16 344 N344Y 0.32 0.91 0.43 0.89 1.49 346 F346A 0.70 1.02 1.25 0.95 0.99 346 F346C 0.48 0.90 0.99 0.82 0.87 346 F346D 0.18 0.26 0.35 1.22 2.13 346 F346E 0.21 0.31 0.30 1.53 2.18 346 F346G 0.60 1.01 0.72 0.97 1.10 346 F346H 1.05 1.02 0.91 1.06 1.22 346 F346I 0.20 0.62 0.31 1.42 1.75 346 F346K 1.01 0.98 0.78 1.00 1.21 346 F346L 0.28 0.66 0.63 0.96 1.13 346 F346M 0.63 0.98 0.77 1.03 1.04 346 F346N 0.43 0.82 0.61 0.92 1.14 346 F346P 0.31 0.24 0.22 1.16 2.49 346 F346Q 0.89 0.93 0.71 1.26 1.70 346 F346R 1.02 0.94 0.69 1.07 1.30 346 F346S 0.78 0.95 0.94 1.01 0.99 346 F346T 0.27 0.85 0.58 0.76 0.98 346 F346V 0.30 0.69 0.60 0.89 1.09 346 F346W 0.41 0.91 0.87 0.96 1.00 346 F346Y 0.91 1.02 0.85 1.10 1.31 356 K356A 1.22 1.03 0.88 0.23 0.25 356 K356C 0.62 0.92 0.98 0.29 0.45 356 K356D 0.42 0.87 0.57 0.22 0.42 356 K356E 0.43 0.98 0.71 0.28 0.43 356 K356F 0.69 0.99 0.45 0.15 0.39 356 K356G 0.97 0.98 0.20 0.20 0.40 356 K356H 0.70 0.88 0.63 0.52 0.52 356 K356I 0.76 0.96 0.60 0.26 0.46 356 K356L 0.67 1.00 0.68 0.45 0.39 356 K356M 0.68 0.99 0.88 0.54 0.53 356 K356N 0.98 1.04 0.72 0.19 0.37 356 K356P 0.23 0.52 0.33 1.09 0.99 356 K356Q 0.77 0.99 0.77 0.41 0.43 356 K356R 0.70 0.98 0.62 0.95 0.76 356 K356S 0.58 0.88 0.48 0.22 0.41 356 K356T 0.63 1.00 0.49 0.28 0.43 356 K356V 0.91 0.90 0.75 0.35 0.31 356 K356W 0.39 0.90 0.42 0.24 0.47 356 K356Y 0.52 0.96 0.47 0.20 0.51 360 G360A 0.56 0.89 0.47 0.08

360 G360E 0.62 0.88 0.45

360 G360F 0.24 0.45 0.08 0.08

360 G360H 0.51 0.84 0.21 0.07

360 G360I 0.41 0.83 0.19 0.07

360 G360K 0.19 0.46 0.07

0.08 360 G360L 0.69 0.93 0.33

360 G360M 0.84 1.06 0.47 0.07 0.05 360 G360N 0.60 0.93 0.23

360 G360Q 0.68 1.01 0.58

360 G360R 0.77 1.01 0.29

360 G360S 0.73 0.96 0.47

360 G360T 0.58 0.97 0.13

360 G360V 0.10 0.07 0.14 4.46 16.21  360 G360W 0.61 0.92 0.27

0.07 360 G360Y 0.11

0.06 0.78 6.25 361 Q361A 0.93 1.02 0.18 0.13 0.14 361 Q361C 0.45 0.91 0.18 0.21 0.20 361 Q361D 0.43 0.65 0.10 0.19 0.29 361 Q361E 0.07 1.37

1.01 0.93 361 Q361F 0.34 0.40

0.31 0.49 361 Q361G 0.33 1.01 0.10 0.23 0.24 361 Q361H 0.25 0.87 0.20 0.74 0.59 361 Q361I 0.19 0.49 0.19 0.91 1.00 361 Q361K 0.66 0.73 0.07 0.27 0.34 361 Q361L 0.86 0.86 0.21 0.20 0.19 361 Q361M 0.59 0.86 0.10 0.27 0.25 361 Q361N 0.06 0.05

0.36 4.82 361 Q361P 0.26 0.21

0.80 0.86 361 Q361R 0.69 0.75 0.10 0.13 0.18 361 Q361S 0.55 0.90 0.13 0.27 0.22 361 Q361T 0.57 0.83 0.12 0.25 0.26 361 Q361V 0.44 0.80 0.09 0.08 0.12 361 Q361W 0.49 0.66 0.27 0.41 0.38 361 Q361Y 0.31 0.39 0.22 0.79 0.58 362 Q362A 0.81 0.98 0.90 0.83 0.69 362 Q362C 0.68 0.95 0.83 0.90 0.98 362 Q362D 1.15 1.02 0.95 0.85 0.78 362 Q362E 0.93 0.95 0.70 0.86 0.81 362 Q362F 0.73 0.97 0.54 0.75 0.66 362 Q362G 0.70 0.99 0.34 0.31 0.44 362 Q362H 1.00 0.96 0.41 0.96 0.96 362 Q362I 0.86 0.99 1.04 1.30 1.81 362 Q362K 0.17 0.44 0.28 0.64 3.22 362 Q362L 0.71 0.96 0.89 1.29 1.86 362 Q362M 0.75 0.96 0.88 1.10 1.45 362 Q362N 0.78 0.98 0.74 0.96 0.99 362 Q362P 0.55 0.86 0.71 0.90 0.90 362 Q362R 0.96 0.96 0.53 1.03 1.15 362 Q362S 0.68 0.98 0.61 0.78 0.80 362 Q362T 0.73 0.99 0.60 0.85 0.73 362 Q362V 1.05 0.96 0.77 0.98 1.00 362 Q362W 0.94 0.97 0.54 0.75 0.60 362 Q362Y 0.78 1.05 0.76 0.71 0.61 363 Q363A 1.26 1.00 0.95 1.07 1.25 363 Q363C 0.27 0.69 0.50 0.50 1.16 363 Q363D 0.30 0.83 0.94 0.98 1.30 363 Q363E 0.91 1.00 0.87 0.97 1.03 363 Q363F 1.37 0.95 0.72 0.56 1.15 363 Q363G 0.17 0.31 0.23 1.14 2.30 363 Q363H 0.39 0.92 0.39 1.05 1.40 363 Q363I 0.43 0.92 0.28 0.31 0.56 363 Q363K 0.44 0.95 0.60 0.63 1.22 363 Q363L 0.21 0.80 0.38 0.93 0.93 363 Q363M 0.65 0.95 0.62 0.90 0.84 363 Q363N 3.15 0.98 1.15 1.31 1.70 363 Q363P 0.37 0.63 0.20 0.48 0.82 363 Q363R 0.94 0.94 0.55 1.11 1.22 363 Q363S 0.62 1.00 0.85 1.05 1.40 363 Q363T 1.10 0.98 0.74 0.99 0.86 363 Q363V 0.18 0.88 0.50 0.86 1.24 363 Q363W 0.70 0.88 0.45 0.68 0.61 363 Q363Y 0.75 0.91 0.75 1.05 1.08 364 W364A 0.95 0.85 0.19 0.13 0.18 364 W364C 0.81 0.69 0.50 0.77 0.65 364 W364D 1.65 0.88 0.31 0.11 0.11 364 W364E 0.69 0.93 0.27 0.18 0.22 364 W364F 0.54 0.77 0.08 0.28 0.30 364 W364G 0.70 0.83 0.21 0.18 0.13 364 W364I 0.46 0.82 0.18 0.14 0.19 364 W364K 0.69 0.50 0.12 0.21 0.09 364 W364L 0.35 0.83 0.13 0.10 0.19 364 W364M 0.63 0.93 0.17 0.19 0.12 364 W364N 0.54 0.83 0.23 0.11 0.11 364 W364P 0.72 0.81 0.08 0.20 0.23 364 W364Q 0.55 0.92 0.16 0.17 0.20 364 W364R 1.01 0.61 0.09 0.14 0.16 364 W364S 0.65 0.82 0.10 0.20 0.24 364 W364T 0.83 0.79 0.11 0.06 0.13 364 W364V 0.52 0.85 0.51 0.12 0.20 364 W364Y 0.66 0.68 0.14 0.21 0.29 365 G365C 0.48 0.59 0.37 0.87 0.88 365 G365E 0.96 0.83 0.57 0.92 0.92 365 G365F 0.38 0.66 0.18 0.64 0.53 365 G365H 0.47 0.72 0.21 0.74 0.66 365 G365I 0.38 0.52 0.09 0.68 0.61 365 G365K 0.74 0.72 0.28 0.85 0.79 365 G365L 0.40 0.57 0.29 0.86 0.86 365 G365M 0.62 0.66 0.39 0.97 1.08 365 G365N 0.30 0.66 0.29 0.84 0.86 365 G365P 0.37 0.64 0.23 0.53 0.26 365 G365Q 0.25 0.15 0.21 0.82 1.38 365 G365R 0.64 0.72 0.26 0.83 0.86 365 G365S 0.71 0.80 0.74 1.01 1.14 365 G365T 0.85 0.66 0.36 0.94 1.11 365 G365V 0.65 0.55 0.17 0.68 0.78 365 G365W 0.80 0.77 0.36 0.66 0.58 365 G365Y 0.06 1.15 0.15 0.99 1.15 371 I371A 0.34 1.05 1.25 0.75 0.57 371 I371C 0.34 0.87 0.84 0.88 0.59 371 I371D 0.62 0.99 0.88 0.90 0.79 371 I371E 0.36 0.98 0.73 0.81 0.54 371 I371F 0.21 0.24 0.07 0.70 0.49 371 I371G 0.54 0.98 0.57 0.88 0.70 371 I371H 0.24 0.08

0.63 0.94 371 I371K 0.11 0.07

0.86 1.24 371 I371L 0.29 0.64 0.55 0.54 0.28 371 I371M 0.28 0.87 0.53 0.68 0.40 371 I371N 0.64 1.06 0.74 0.93 0.82 371 I371P 0.21 0.18

1.78 1.31 371 I371Q 0.31 0.81 0.67 0.77 0.57 371 I371R 0.21 0.43 0.13 0.63 0.55 371 I371S 1.05 1.00 1.10 1.14 1.12 371 I371T 0.95 1.00 0.80 1.08 1.00 371 I371V 0.28 0.59 0.37 0.73 0.52 371 I371W 0.23 0.18 0.10 0.74 0.73 371 I371Y 0.24 0.42 0.27 0.56 0.45 378 R378A 0.21 0.76 0.79 0.25 0.54 378 R378C 0.25 0.34 0.42 0.74 1.01 378 R378D 0.19 0.23 0.33 0.49 0.58 378 R378E 0.17 0.18 0.30 0.92 0.94 378 R378F 0.13 0.39 1.34 0.12 0.38 378 R378G 0.08 0.22 1.48 0.19 0.82 378 R378H 0.13 0.22 0.82 0.62 1.22 378 R378I 0.19 0.42 0.33 0.38 0.69 378 R378K 0.15 0.45 0.27 0.61 0.98 378 R378L 0.21 0.75 0.79 0.33 0.53 378 R378M 0.14 0.52 0.77 0.37 0.47 378 R378N 0.12 0.19 0.48 0.38 0.81 378 R378P 0.17 0.65 0.54 0.48 0.62 378 R378Q 0.20 0.82 0.94 0.56 0.59 378 R378S 0.18 0.65 0.47 0.45 0.64 378 R378T 0.18 0.52 1.03 0.25 0.43 378 R378V 0.36 1.01 0.98 1.06 1.19 378 R378W 0.09 0.10 0.64 0.34 0.30 378 R378Y 0.24 0.60 0.62 0.23 0.53 380 S380A 0.95 0.99 0.48 0.31 0.10 380 S380C 0.52 1.03 0.63 0.57 0.37 380 S380D 0.61 1.12 0.45 0.12 0.16 380 S380E 0.52 0.94 0.29 0.41 0.20 380 S380F 0.60 1.05 0.22 0.08 0.10 380 S380G 0.68 1.05 0.20 0.17 0.21 380 S380H 0.68 1.09 0.37 0.42 0.14 380 S380I 0.73 1.06 0.66 0.99 0.96 380 S380K 0.80 1.08 0.42 0.94 0.94 380 S380L 1.04 1.07 0.57 0.78 0.60 380 S380M 1.02 1.03 0.43 0.85 0.69 380 S380N 0.81 1.09 0.58 0.95 0.89 380 S380P 0.73 1.04 0.08 0.07 0.11 380 S380Q 0.69 1.02 0.44 0.45 0.17 380 S380R 0.83 1.04 0.38 0.86 0.73 380 S380T 0.30 0.70 0.17 0.56 0.35 380 S380V 0.98 1.02 0.79 0.99 0.96 380 S380W 0.85 1.00 0.11

0.06 380 S380Y 1.01 1.01 0.25 0.14 0.10 381 A381C 0.67 1.05 0.94 0.89 0.83 381 A381D 0.67 1.05 0.96 0.90 0.79 381 A381E 0.52 1.08 0.84 0.85 0.75 381 A381F 0.69 1.07 0.66 0.89 0.82 381 A381G 0.36 0.98 0.76 0.76 0.65 381 A381H 0.14 0.15

1.39 0.95 381 A381I 1.12 1.09 0.84 0.89 0.83 381 A381K 0.13 0.11

0.27 1.00 381 A381L 0.52 0.95 0.62 0.78 0.62 381 A381M 0.47 1.05 0.68 0.82 0.75 381 A381N 0.38 0.89 0.62 0.84 0.76 381 A381P 0.54 1.00 0.72 0.94 1.02 381 A381Q 0.48 1.01 0.60 0.87 0.75 381 A381R 0.39 0.95 0.59 0.80 0.75 381 A381S 1.20 1.05 0.78 1.04 1.06 381 A381T 0.93 0.97 0.73 0.97 1.01 381 A381V 0.54 0.97 0.59 0.90 0.81 381 A381W 0.54 0.87 0.64 0.97 0.93 381 A381Y 0.61 0.92 0.60 0.92 0.87 382 N382A 1.22 0.70 2.07 0.91 382 N382C 1.11 0.57 1.52 1.54 382 N382D 0.71 0.81 1.17 1.15 382 N382E 2.21 0.16 6.19 5.56 382 N382F 0.91 1.23 1.03 0.66 382 N382G 0.78 0.56 1.73 1.31 382 N382H 0.81 1.01 1.23 1.08 382 N382I 0.48 0.80 1.63 1.35 382 N382K 1.66 0.50 2.10 1.85 382 N382L 0.56 0.74 1.58 1.31 382 N382M 0.50 0.66 1.75 1.57 382 N382Q 1.08 0.65 1.45 1.46 382 N382T 0.50 1.04 1.31 0.99 382 N382V 1.53 0.58 1.57 1.66 382 N382Y 0.49 0.62 1.67 1.55 384 G384A 0.58 0.72 0.68 1.26 0.79 384 G384C 0.65 1.06 1.22 0.88 0.91 384 G384D 1.36 1.06 1.31 0.84 0.73 384 G384E 0.52 0.92 1.05 1.33 0.96 384 G384F 0.75 1.00 0.87 1.16 0.96 384 G384H 0.48 0.73 0.86 1.02 1.01 384 G384K 0.50 0.60 1.41 1.56 384 G384L 0.41 0.76 1.03 0.95 0.90 384 G384M 0.61 0.70 1.01 1.19 1.24 384 G384N 0.76 0.53 0.61 1.03 1.43 384 G384Q 0.57 1.15 1.19 0.92 0.85 384 G384R 0.67 0.62 0.55 1.20 1.70 384 G384S 0.16 0.17 0.51 0.59 1.21 384 G384T 0.87 0.06 4.44 3.08 384 G384V 0.38 0.80 0.53 0.72 0.80 384 G384W 0.69 1.12 0.71 0.97 0.95 384 G384Y 0.45 0.50 1.83 2.73 386 S386A 1.89 2.69 3.12 0.99 1.88 386 S386C 2.63 2.50 3.75 1.03 2.09 386 S386D 1.57 2.52 2.56 0.92 1.79 386 S386E 6.64 2.19 4.12 1.05 2.33 386 S386F 2.11 3.11 2.96 1.05 2.19 386 S386G 0.45 0.51 0.43 1.78 3.74 386 S386H 2.52 2.56 2.50 1.11 2.13 386 S386I 2.44 2.52 2.64 1.08 2.03 386 S386K 0.93 0.36 0.30 2.91 2.66 386 S386L 1.33 2.29 1.43 0.92 1.65 386 S386M 0.43 0.26

1.20 7.13 386 S386P 4.61 2.34 2.31 1.10 2.27 386 S386Q 0.57 2.58 0.27 0.38 0.71 386 S386R 1.16 2.27 0.76 0.91 1.58 386 S386T 1.05 2.01 1.88 0.96 1.70 386 S386V 3.71 2.47 3.11 1.04 2.15 386 S386W 0.88 2.31 1.52 0.97 1.32 394 V394A 0.53 0.99 0.74 0.67 0.40 394 V394C 0.32 0.87 0.81 0.80 0.52 394 V394D 0.11 0.18 0.31 0.98 0.85 394 V394E 0.06 0.43

0.34 0.64 394 V394F 0.06 0.42 0.17 0.46 0.56 394 V394G 0.27 0.76 0.56 0.48 0.28 394 V394H 0.09 0.18 0.07 0.68 1.18 394 V394I 0.51 1.01 0.76 0.89 0.80 394 V394K 0.07 0.23 0.73 1.04 0.86 394 V394L 0.17 0.87 0.23 0.09 0.24 394 V394M 0.71 0.94 0.31 0.11 0.11 394 V394N 0.09 0.30 0.18 0.79 0.67 394 V394P 0.11 0.18 0.08 0.73 1.42 394 V394Q 0.11 0.22 0.11 0.87 1.30 394 V394R 0.06 0.28 0.23 0.87 1.11 394 V394S 0.12 0.58 0.19 0.21 0.37 394 V394T 0.22 0.79 0.29 0.15 0.21 394 V394W 0.09 0.15 0.31 0.15 1.55 394 V394Y 0.11 0.17

0.54 1.12 396 P396A 0.52 0.68 1.01 0.81 0.51 396 P396C 0.29 0.24 0.73 0.76 0.73 396 P396D 0.09 0.56 0.88 0.44 0.76 396 P396E 0.31 0.58 0.90 0.48 0.44 396 P396F 0.29 0.48 0.48 0.29 0.30 396 P396G 0.49 0.63 0.82 0.44 0.25 396 P396H 0.19 0.33 0.58 0.50 0.75 396 P396I 0.34 0.57 0.52 0.11 0.35 396 P396K 0.34 0.29 0.40 0.43 0.80 396 P396L 0.48 0.51 0.52 0.16 0.30 396 P396M 0.40 0.66 0.52 0.12 0.31 396 P396N 0.12 0.47 0.58 0.79 0.98 396 P396Q 0.06

0.32 0.65 1.26 396 P396R 0.29 0.31 0.46 0.24 0.56 396 P396S 0.16 0.14 0.43 0.35 1.18 396 P396T 0.48 0.60 0.50 0.22 0.26 396 P396V 0.40 0.59 0.51 0.42 0.27 396 P396W 0.26 0.25 0.56 0.21 0.30 396 P396Y 0.27 0.32 0.42 0.37 0.37 399 E399A 0.68 0.36 0.39 0.09 0.08 399 E399C 0.31 0.11 0.26 0.24 0.19 399 E399D 0.15 0.31 0.21 0.32 0.39 399 E399F 1.02 0.30 0.25 0.11

399 E399G 0.75 0.32 0.20 0.13

399 E399H 0.86 0.29 0.33 0.18 0.16 399 E399I 0.54 0.18 0.16 0.80 0.33 399 E399K 0.43 0.21 0.21 0.35 0.22 399 E399L 0.68 0.19 0.23 0.32 0.24 399 E399M 0.56 0.22 0.21 0.31 0.28 399 E399N 0.50 0.33 0.33 0.20 0.19 399 E399P 0.80 0.23 0.27 0.15 0.17 399 E399Q 0.69 0.68 0.40 0.30 0.09 399 E399R 0.69 0.13 0.15 0.62 0.49 399 E399S 0.64 0.39 0.23 0.13 0.14 399 E399T 0.12 0.31

1.16 1.26 399 E399V 0.49 0.20 0.25 0.53 0.54 399 E399W 1.00 0.30 0.31 0.55 0.30 399 E399Y 0.82 0.30 0.26 0.22 0.19 400 C400A 1.11 0.98 0.72 0.89 0.74 400 C400D 0.42 0.76 0.48 0.48 0.48 400 C400E 0.32 0.35

0.47 0.28 400 C400F 0.57 0.27

0.50 0.22 400 C400G 0.75 1.01 0.47 0.32 0.11 400 C400H 0.24 0.11 0.09 1.53 1.07 400 C400I 0.65 0.40 0.10 0.51 0.11 400 C400K 0.63 0.36 0.08 0.32 0.33 400 C400L 0.62 0.36

0.60 0.49 400 C400M 0.69 0.46

0.22 0.29 400 C400N 0.11

0.09 5.30 3.48 400 C400P 0.48 0.38 0.05 0.45 0.36 400 C400Q 0.45 0.21 0.05 0.87 0.61 400 C400R 0.61 0.20 0.07 0.57 0.73 400 C400S 0.88 0.90 1.02 1.04 1.51 400 C400T 0.93 0.75 0.14 0.20 0.21 400 C400V 1.18 0.55 0.25 0.23 0.14 400 C400W 0.32 0.09 0.08 0.27 0.22 400 C400Y 0.57 0.20

0.40 0.22 405 D405A 1.75 0.82 0.33 0.32 0.60 405 D405C 0.45 0.63 0.57 0.72 0.97 405 D405E 0.85 0.81 0.63 0.50 0.65 405 D405F 0.79 0.95 0.24 0.56 0.82 405 D405G 0.77 0.87 0.16 0.56 0.86 405 D405H 0.27 0.38 0.25 3.35 3.59 405 D405I 0.62 1.04 0.49 0.83 1.02 405 D405K 0.63 0.65 0.24 0.43 0.72 405 D405L 0.41 0.95 0.76 0.38 0.43 405 D405M 0.52 1.31 0.58 0.59 0.69 405 D405N 0.74 1.04 0.78 1.34 1.32 405 D405P 0.77 0.80 0.19 0.99 1.02 405 D405Q 0.71 1.12 0.39 1.10 1.21 405 D405R 0.93 1.20 0.36 0.56 0.61 405 D405S 0.95 1.24 0.40 0.75 0.96 405 D405T 0.74 1.17 0.32 0.64 0.83 405 D405V 0.39 1.05 0.60 1.32 1.24 405 D405W 1.12 1.17 0.40 0.42 0.48 405 D405Y 1.56 1.07 0.39 0.31 0.07 406 S406A 1.28 1.05 1.70 1.01 0.99 406 S406C 0.57 0.86 1.05 0.66 0.71 406 S406D 0.10 0.09 0.07 0.34 1.33 406 S406E 1.24 1.04 0.99 0.94 0.90 406 S406F 1.01 1.04 0.72 0.56 0.38 406 S406G 0.84 1.00 0.67 0.49 0.39 406 S406H 0.62 0.99 0.78 0.85 0.79 406 S406I 2.11 1.06 0.70 0.83 0.63 406 S406K 0.15 0.11

1.18 0.67 406 S406L 0.18 0.22 0.32 0.26 0.42 406 S406M 0.89 1.01 0.78 0.85 0.75 406 S406N 0.73 1.02 0.79 0.80 0.63 406 S406P 1.07 1.02 1.10 1.16 1.41 406 S406Q 1.01 0.99 0.77 0.90 0.79 406 S406R 1.26 0.99 0.73 1.13 1.28 406 S406T 0.83 0.98 0.77 0.80 0.72 406 S406V 0.95 1.00 0.61 0.76 0.57 406 S406W 0.72 0.96 1.15 1.03 1.21 406 S406Y 0.86 0.98 0.77 0.59 0.39 407 S407A 0.92 1.00 1.15 0.89 0.86 407 S407C 0.55 1.03 1.19 0.80 0.87 407 S407D 0.93 1.00 0.91 0.71 0.61 407 S407E 0.79 1.00 1.03 0.83 0.75 407 S407F 1.19 1.05 0.57 0.61 0.39 407 S407G 0.90 1.03 0.63 0.80 0.63 407 S407H 1.17 1.01 0.61 1.09 1.27 407 S407I 0.23 0.13 0.21 0.57 1.16 407 S407K 0.90 1.04 0.49 1.07 1.29 407 S407L 1.02 1.08 0.66 0.73 0.56 407 S407M 0.11 0.13 0.09 0.37 1.24 407 S407N 1.09 1.02 0.70 1.06 1.12 407 S407P 0.56 0.94 0.64 0.19 0.39 407 S407Q 0.09 0.13 0.28 0.40 1.29 407 S407R 1.07 1.02 0.67 1.08 1.17 407 S407T 1.26 1.06 0.98 1.11 1.21 407 S407V 0.91 0.99 0.39 0.38 0.38 407 S407W 0.79 0.99 0.47 0.37 0.41 407 S407Y 1.01 0.99 0.49 0.55 0.45 410 R410A 0.84 0.71 0.52 0.99 1.09 410 R410C 0.43 0.74 0.99 1.09 1.15 410 R410D 1.27 0.78 0.90 1.21 1.52 410 R410E 0.65 0.74 0.47 1.10 1.21 410 R410F 0.54 0.65 0.43 1.04 1.23 410 R410G 0.69 0.73 0.67 1.19 1.61 410 R410H 0.64 0.74 0.85 1.18 1.60 410 R410I 0.49 0.53 0.38 0.77 0.82 410 R410K 0.73 0.76 0.90 1.23 1.20 410 R410L 0.89 0.73 0.65 0.87 0.84 410 R410M 0.53 0.71 0.70 0.90 1.03 410 R410N 0.77 0.76 0.97 1.22 1.58 410 R410P 0.49 0.56 0.42 0.45 0.75 410 R410Q 0.70 0.78 0.86 1.05 1.30 410 R410S 0.63 1.51 1.83 1.14 1.38 410 R410T 0.68 0.61 0.57 1.00 1.29 410 R410V 0.57 0.58 0.56 0.87 1.19 410 R410W 0.72 0.66 0.36 0.84 0.86 410 R410Y 0.71 0.63 0.68 1.11 1.40 413 S413A 0.57 1.15 1.04 1.21 1.01 413 S413C 0.58 1.03 0.72 0.98 1.05 413 S413D 0.28 0.90 0.90 0.64 0.72 413 S413E 0.48 0.89 0.51 0.83 1.22 413 S413F 0.96 0.80 0.61 1.71 1.87 413 S413G 0.35 0.81 0.61 1.26 1.07 413 S413H 0.71 0.81 0.45 1.10 1.02 413 S413I 0.70 1.18 0.72 1.06 1.23 413 S413K 0.64 0.86 0.61 0.99 0.82 413 S413L 1.00 1.05 0.72 1.10 1.46 413 S413M 0.83 0.87 0.59 1.31 1.48 413 S413N 0.54 1.03 0.81 0.73 0.69 413 S413P 0.27 0.76 0.62 1.71 2.11 413 S413Q 0.45 1.03 0.89 0.96 1.07 413 S413R 0.58 1.07 0.56 1.12 0.90 413 S413T 0.63 1.08 0.85 0.92 0.87 413 S413V 1.18 0.98 0.92 0.98 1.26 413 S413W 0.40 0.96 0.78 1.61 1.47 413 S413Y 0.71 0.88 0.48 1.68 1.49 414 H414A 1.38 0.98 0.99 1.04 1.08 414 H414C 0.25

0.37 4.50 4.02 414 H414D 0.85 0.82 0.92 1.01 0.88 414 H414E 0.50 0.91 0.43 0.05 0.11 414 H414F 0.78 0.97 0.73 0.86 0.57 414 H414G 0.97 0.89 0.61 0.51 0.20 414 H414I 0.57 0.94 0.56 0.34 0.20 414 H414K 0.82 1.04 0.94 0.89 0.77 414 H414L 0.78 0.93 0.45 0.27 0.17 414 H414M 0.46 0.97 1.09 0.95 1.06 414 H414N 0.10 0.13 0.13 1.62 2.06 414 H414P 0.21 0.05

3.19 0.30 414 H414Q 0.73 0.95 0.72 0.78 0.64 414 H414R 0.22 0.07 0.09 1.93 2.69 414 H414S 0.67 0.92 0.93 0.83 0.84 414 H414T 0.61 1.01 0.72 0.76 0.50 414 H414V 0.54 0.90 0.65 0.61 0.38 414 H414W 0.28 0.48 0.47 0.58 0.24 414 H414Y 1.00 0.98 0.78 0.86 0.67 416 A416C 0.49 0.73 0.49 0.58 0.23 416 A416D 0.25 0.47 0.38 0.16

416 A416E 0.37 0.98 0.69 0.34

416 A416F 0.38 0.97 0.28

0.10 416 A416G 0.49 0.61 0.40 0.52 0.25 416 A416H 0.43 0.87 0.23 0.10

416 A416I 0.59 0.98 0.68 0.67 0.29 416 A416K 0.58 0.99 0.48 0.61 0.41 416 A416L 0.22 0.46 0.12 0.19 0.17 416 A416M 0.32 0.92 0.49 0.42 0.19 416 A416P 0.27 0.30 0.12 0.14

416 A416Q 0.37 0.98 0.50 0.56 0.33 416 A416R 0.58 0.95 0.20 0.59 0.31 416 A416S 0.24 0.77 0.62 0.68 0.58 416 A416T 0.58 0.98 0.68 0.76 0.59 416 A416V 0.59 1.10 0.56 0.66 0.33 416 A416W 0.42

0.15 0.50 1.05 416 A416Y 0.38 0.84 0.17 0.07

417 L417A 0.51 0.95 0.90 0.75 0.50 417 L417C 0.36 0.81 0.57 0.35

417 L417D 0.49 0.90 0.49 0.10

417 L417E 0.22 0.62 0.50 0.36 0.11 417 L417F 0.45 0.96 0.74 0.67 0.49 417 L417G 0.57 0.99 0.42 0.54 0.21 417 L417H 0.76 1.02 0.24 0.12 0.10 417 L417I 0.48 0.89 0.30 0.20

417 L417K 0.51 1.01 0.59 0.82 0.81 417 L417M 0.53 1.02 0.73 0.78 0.59 417 L417P 0.23 0.62 0.28 0.07 0.07 417 L417R 0.81 1.01 0.46 0.80 0.68 417 L417S 0.80 1.01 0.74 0.61 0.39 417 L417T 0.66 1.01 0.51 0.41 0.12 417 L417V 0.50 0.89 0.45 0.23

417 L417W 0.80 0.98 0.28

417 L417Y 0.33 0.70 0.58 0.80 0.48 422 Q422A 1.39 0.93 0.75 0.66 0.68 422 Q422C 0.84 0.94 0.40 1.04 0.86 422 Q422D 0.31 1.58

0.43 0.90 422 Q422E 0.95

53.56  10.85  422 Q422G 0.33 1.32 0.10 0.89 1.23 422 Q422H 1.35 0.75 0.09 0.96 1.14 422 Q422I 0.93 1.12 0.60 0.47 0.87 422 Q422K 0.64 1.08 0.55 1.07 1.09 422 Q422L 0.93 0.89 0.20 1.13 0.94 422 Q422M 0.41 0.83 0.43 2.12 1.83 422 Q422N 1.12 0.37

2.24 2.42 422 Q422P 0.33 1.01 0.07 1.52 1.20 422 Q422R 0.64 0.93 0.35 1.34 1.04 422 Q422S 0.58 1.20 0.19 0.79 0.75 422 Q422T 0.57 1.22 0.91 0.73 0.93 422 Q422V 0.51 1.27 0.94 1.17 0.97 422 Q422W 0.32

0.11 6.62 0.06 422 Q422Y 0.71 0.70 0.11 1.26 1.39 426 Q426A 0.30 0.53 0.69 0.68 0.57 426 Q426C 0.83 0.83 0.81 0.78 0.78 426 Q426D 0.90 0.93 1.02 0.97 0.91 426 Q426E 0.05 0.36 0.20 1.40 1.97 426 Q426F 0.47 0.76 0.56 0.58 0.31 426 Q426G 0.75 0.82 0.69 0.87 0.66 426 Q426H 0.82 0.81 0.46 0.70 0.38 426 Q426I 0.59 0.72 0.75 1.01 0.89 426 Q426K 0.63 0.65 0.44 1.29 1.23 426 Q426L 0.86 0.87 0.56 0.74 0.54 426 Q426M 0.52 0.71 0.67 0.96 1.00 426 Q426N 0.51 0.77 0.49 1.06 0.98 426 Q426P 0.60 0.85 0.76 0.98 1.02 426 Q426R 0.51 0.87 0.43 0.79 0.79 426 Q426S 0.67 0.84 0.75 0.88 0.73 426 Q426T 0.63 0.88 0.71 0.81 0.64 426 Q426V 0.51 0.91 0.44 0.53 0.40 426 Q426W 0.38 0.72 0.44 0.49 0.23 426 Q426Y 0.44 0.72 0.64 0.67 0.33 427 A427C 1.03 0.36 0.31 0.88 0.54 427 A427D 1.06 0.48 0.31 0.16 0.23 427 A427F 0.65 0.80 0.52 1.08 0.97 427 A427G 0.84 0.78 0.44 0.37 0.29 427 A427H 0.92 0.39 0.14 0.59 0.36 427 A427I 0.92 0.33 0.14 0.62 0.50 427 A427L 0.62 0.20 0.16 0.76 0.40 427 A427M 0.70 0.12 0.08 1.76 1.63 427 A427N 1.34 0.77 0.42 0.36 0.16 427 A427P 0.79 0.59 0.35 0.60 0.51 427 A427Q 0.14 0.10

10.42  3.80 427 A427R 0.96 0.16 0.23 1.03 1.68 427 A427S 0.88 1.00 0.47 0.66 0.46 427 A427T 0.67 0.75 0.61 0.58 0.47 427 A427V 1.14 0.59 0.46 0.83 0.61 427 A427W 0.95 0.07 0.08 1.67 2.16 427 A427Y 0.11 0.43

0.40 0.97 429 A429C 0.39 0.69 0.69 0.88 0.82 429 A429D 0.70 0.88 1.07 1.07 0.90 429 A429E 1.11 0.90 0.90 1.10 1.11 429 A429F 0.54 1.08 0.65 0.72 0.45 429 A429G 0.62 0.88 0.61 0.66 0.41 429 A429H 0.41 0.76 0.26 0.19 0.41 429 A429I 0.59 0.92 0.55 0.61 0.34 429 A429K 0.89 1.03 0.69 0.83 0.84 429 A429L 0.46 0.91 0.74 0.92 0.67 429 A429M 0.99 1.07 1.05 0.93 0.97 429 A429N 0.13 0.18 0.22 0.38 1.99 429 A429P 0.46 0.48 0.24 0.17 0.48 429 A429Q 0.36 1.10 0.60 0.82 0.73 429 A429R 0.75 1.04 0.54 0.87 0.75 429 A429S 1.34 1.04 0.85 0.90 0.84 429 A429T 0.66 1.03 0.74 0.87 0.81 429 A429V 0.20 0.32 0.42 0.53 0.53 429 A429W 0.29 0.87 0.32

0.16 429 A429Y 0.66 1.04 0.74 0.76 0.61 431 F431A 0.40 0.97 0.35 0.22 0.18 431 F431C 0.10 3.60 0.09 1.05 1.36 431 F431D 0.52 0.94 0.41 0.13 0.14 431 F431E 0.33 0.59 0.37 0.23 0.28 431 F431G 0.33 0.37 0.61 0.44 0.59 431 F431H 0.71 0.91 0.43 0.17 0.21 431 F431I 0.93 0.92 0.36 0.06 0.18 431 F431K 0.25 0.22 0.29 0.99 1.10 431 F431L 0.61 1.04 0.32 0.09 0.13 431 F431M 0.46 0.94 0.53 0.12 0.17 431 F431P 0.16 0.20 0.36 1.11 1.82 431 F431Q 0.33 1.06 0.45 0.12 0.26 431 F431S 0.30 0.97 0.28 0.19 0.27 431 F431T 0.10 1.39 0.30 0.27 0.36 431 F431V 0.38 1.04 0.58 0.20 0.18 431 F431W 0.36 0.92 0.15 0.10 0.22 431 F431Y 0.57 0.98 0.40 0.48 0.14 433 A433C 0.42 0.96 0.69 0.55 0.33 433 A433D 1.23 1.06 0.93 0.78 0.63 433 A433E 0.35 1.00

0.10 0.15 433 A433F 0.57 1.01 0.13 0.10 0.09 433 A433G 1.08 1.08 0.84 0.56 0.25 433 A433H 0.10 0.09

1.01 4.13 433 A433I 0.40 0.98 0.49 0.27 0.10 433 A433K 0.53 0.98 0.70 0.82 0.66 433 A433L 0.46 1.07 0.57 0.71 0.51 433 A433M 0.70 1.00 0.72 0.71 0.54 433 A433N 0.60 0.93 0.86 0.69 0.49 433 A433P 0.52 1.00 0.66 0.71 0.50 433 A433Q 0.85 1.06 0.71 0.81 0.68 433 A433R 1.25 1.00 0.82 0.85 0.67 433 A433S 0.81 1.03 0.70 0.72 0.54 433 A433T 0.57 1.01 0.65 0.50 0.28 433 A433V 0.46 0.94 0.90 0.21 0.14 433 A433W 0.28 0.55 0.28 0.16 0.20 433 A433Y 0.59 0.90 0.52 0.08 0.09 436 V436A 0.87 1.02 0.91 0.41 0.17 436 V436C 0.73 0.96 0.76 0.73 0.48 436 V436D 0.98 0.99 0.77 0.35 0.14 436 V436E 1.11 1.04 0.75 0.76 0.58 436 V436F 0.64 0.99 0.53 0.45 0.15 436 V436G 0.37 0.93 0.26 0.15 0.20 436 V436H 0.55 0.99 0.45 0.10 0.09 436 V436I 0.58 1.02 0.76 0.93 0.87 436 V436K 0.29 0.67 0.15 0.35 0.29 436 V436L 0.66 1.02 0.77 0.80 0.68 436 V436M 0.53 0.99 0.74 0.69 0.42 436 V436N 0.30 0.76 0.31 0.29 0.14 436 V436P 0.28 0.18

0.59 0.99 436 V436Q 0.53 0.90 0.49 0.47 0.20 436 V436R 0.87 0.98 0.47 0.28 0.10 436 V436S 0.47 0.49 0.13 0.28 0.14 436 V436T 0.74 1.03 0.68 0.53 0.26 436 V436W 0.59 0.94 0.56 0.11 0.13 436 V436Y

440 T440A 1.17 1.02 0.84 0.99 0.92 440 T440C 0.60 0.93 0.92 1.08 1.07 440 T440D 0.86 0.97 0.85 0.90 0.79 440 T440E 0.77 1.01 0.85 1.04 1.12 440 T440F 0.65 1.01 0.50 0.91 0.82 440 T440G 0.40 0.91 0.51 0.66 0.51 440 T440H 1.01 1.06 0.52 0.85 0.80 440 T440I 0.43 1.00 0.68 1.07 1.12 440 T440K 0.82 1.02 0.64 1.01 1.07 440 T440L 0.56 0.75 0.48 1.06 1.08 440 T440M 0.62 1.03 0.72 1.02 1.06 440 T440N 0.68 0.91 0.24 0.11 0.13 440 T440P 0.25 0.26 0.10 0.72 0.77 440 T440Q 1.26 1.02 0.82 1.05 1.12 440 T440R 0.40 0.73 0.15 0.86 0.89 440 T440S 0.88 1.01 0.91 0.94 0.93 440 T440V 0.27 0.40

0.28 0.27 440 T440W 0.78 0.89 0.47 0.95 0.90 440 T440Y 1.22 1.01 0.93 0.90 0.91 441 N441A 0.16 0.07 0.71 0.26 1.16 441 N441C 0.28 0.78 1.16 0.27 0.12 441 N441D 0.09

0.21 1.89 3.43 441 N441E 0.48 0.89 0.96 0.44 0.10 441 N441F 0.42 0.86 0.91 0.88 0.61 441 N441G 0.42 0.87 0.79 0.33 0.09 441 N441H 0.11 0.06 0.15 3.38 1.88 441 N441I 0.17 0.39 0.52 0.10 0.10 441 N441K 0.29 0.84 0.72 0.30 0.21 441 N441L 0.46 0.89 0.92 0.83 0.67 441 N441M 0.38 0.87 0.66 0.41 0.10 441 N441P 0.20 0.37 0.40 0.17 0.28 441 N441Q 0.37 0.90 0.71 0.28 0.14 441 N441R 0.28 0.81 0.57 0.26 0.18 441 N441S 0.39 0.89 0.88 0.52 0.31 441 N441T 0.26 0.70 0.55

0.14 441 N441V 0.18 0.55 0.49 0.15 0.23 441 N441W 0.31 0.79 0.72 0.70 0.36 441 N441Y 0.33 0.83 0.88 0.80 0.68 443 N443C 0.34 1.74 1.69 1.22 0.94 443 N443D 0.24 1.85 1.21 1.01 0.73 443 N443E 0.30 1.75 0.76 1.19 0.89 443 N443F 0.79 0.68 0.42 1.14 1.02 443 N443G 0.26 1.00 0.23 1.15 1.43 443 N443I 0.38 1.07 0.53 1.40 1.20 443 N443L 0.83 0.37 0.40 1.88 2.31 443 N443M 0.49 0.96 0.62 1.13 1.29 443 N443P 0.48 0.16 0.13 1.56 4.87 443 N443Q 0.34 1.36 0.89 0.46 1.02 443 N443R 0.31 1.34 0.40 0.50 0.91 443 N443S 0.51 1.21 0.77 0.93 0.76 443 N443T 0.54 0.33 0.12 3.61 2.60 443 N443V 0.47 1.22 0.43 1.20 0.86 443 N443W 0.97

0.10 2.57 0.94 443 N443Y 0.60

0.33

444 P444A 0.85 0.40 0.34 2.29 1.44 444 P444C 0.42 0.61 0.74 2.47 2.22 444 P444D 2.18 0.53 0.54 0.82 1.78 444 P444E 0.93 0.53 0.29 12.61  4.67 444 P444F 0.42 0.58 0.52 3.38 2.94 444 P444G 0.48 0.53 0.42 2.38 1.50 444 P444H 0.27 0.63 0.25 1.19 1.66 444 P444I 0.25 0.54 0.40 1.39 1.77 444 P444K 0.53 0.47 0.51 1.53 1.73 444 P444L 0.20 0.58 0.45 1.57 1.57 444 P444M 0.33 0.39 0.45 2.24 2.88 444 P444N 0.33 0.42 0.43 1.63 1.88 444 P444Q 0.40 0.66 0.49 1.74 1.66 444 P444R 0.38 0.43 0.54 1.98 1.85 444 P444S 0.98 0.49 0.33 1.47 2.17 444 P444T 0.78 0.65 0.52 0.68 1.08 444 P444V 0.33 0.54 0.41 1.80 1.60 444 P444W 0.75 0.49 0.31 1.85 2.13 444 P444Y 0.33 0.84 0.59 0.50 0.69 445 S445A 0.27 0.68 0.52 0.85 0.73 445 S445D 0.35 0.88 0.90 0.57 0.22 445 S445E 0.49 0.95 0.74 0.75 0.54 445 S445F 0.40 0.54 0.33 0.24 0.17 445 S445G 0.37 0.67 0.61 0.76 0.64 445 S445H 0.07 0.16

0.71 1.15 445 S445I 0.41 0.61 0.34 0.34 0.20 445 S445L 0.51 0.68 0.35 0.36 0.27 445 S445N 0.58 1.03 0.77 0.72 0.51 445 S445P 0.65 1.00 0.67 1.01 1.09 445 S445Q 0.32 0.83 0.57 0.78 0.60 445 S445R 0.75 1.01 0.85 0.86 0.83 445 S445T 0.41 0.90 0.74 0.85 0.82 445 S445V 0.53 0.83 0.62 0.57 0.38 445 S445W 0.32 0.55 0.14 0.17 0.26 445 S445Y 0.33 0.70 0.29 0.27 0.13 447 L447A 0.46 0.92 0.97 0.67 0.39 447 L447C 0.47 0.94 0.96 0.84 0.70 447 L447D 0.51 0.96 1.27 0.64 0.40 447 L447E 0.45 0.92 0.98 0.77 0.57 447 L447F 0.40 0.91 0.92 0.99 1.10 447 L447G 0.37 0.83 0.94 0.71 0.42 447 L447H 0.11 0.09 0.14 1.66 2.62 447 L447I 0.29 0.39 0.37 0.57 0.59 447 L447K 0.34 0.93 0.94 0.63 0.45 447 L447M 0.76 1.02 0.99 0.93 0.97 447 L447N 0.58 0.97 0.86 0.80 0.52 447 L447P 0.44 0.97 0.98 0.74 0.50 447 L447Q 0.49 0.97 0.68 0.72 0.51 447 L447R 0.41 0.95 1.02 0.64 0.43 447 L447S 0.50 0.95 0.59 0.70 0.42 447 L447T 0.51 0.99 0.93 0.78 0.59 447 L447V 0.72 0.99 1.02 0.86 0.76 447 L447W 0.60 0.96 0.85 0.89 0.79 447 L447Y 0.69 0.97 0.89 0.92 0.89

Non Combinable variants are those for which all Pi values are ≦0.05. For Hypocrea jecorina CBH2, of the 2,828 variants, 6 are non-combinable. They are A121Q, L243H, A322Q, L328C, L328Y, V436Y. Any CBH2 which has one of the above substitutions relative to Hypocrea jecorina CBH2 can be improved by mutating that amino acid to one of the combinable substitutions at that position, or to the amino acid present in Hypocrea jecorina CBH2 at position 121, 243, 322, 328, or 463.

TABLE 3-2 Act. Act. Act. EtOH Heat CBH2 Combinatorial Variant HPLC CC PASC PCS ratio ratio CBH2.L111S/L144W/T154C/Y162N 0.17 0.61 0.67 1.45 1.05 1.16 CBH2.L111S/L144W/T154V/Y162N/S413Y 0.39 0.39 0.73 0.97 0.97 1.05 CBH2.L144W/T154C/S413Y 0.39 0.32 0.88 1.07 1.02 1.09 CBH2.L111S/T154V 0.10 0.72 0.49 0.92 1.06 1.12 CBH2.T154C 0.47 0.44 1.02 1.05 0.92 0.89 CBH2.L144Q/T154V/Y162N/R410S/S413Y 0.15 0.31 0.50 0.81 0.95 0.96 CBH2.L111S/T154C/Y162N 0.13 5.44 1.11 6.50 0.91 0.82 CBH2.L111S/L144W/Y162N/R410S/S413W 0.12 1.03 0.50 0.94 0.97 1.07 CBH2.L111S/L144W/T154C/R410S/S413Y 0.08 0.79 0.48 0.85 0.88 0.85 CBH2.L144W/T154C/Y162N/R410S/S413W 0.12 1.52 0.43 0.23 0.95 0.55 CBH2.Y162N/R410S/S413Y 0.57 0.55 0.77 0.89 1.09 1.42 CBH2.L144W/T154C/R410S/S413Y 0.52 0.72 0.85 0.97 1.05 1.32 CBH2.L144Q 0.61 0.41 0.75 0.98 1.10 1.39 CBH2.L111S/L144W/R410S/S413Y 0.66 0.46 0.70 0.67 1.11 1.43 No Sequence 0.20 0.23 0.54 1.06 1.07 1.19 CBH2.L144Q/Y162N/R410S/S413Y 0.20 0.62 0.69 0.84 1.02 1.42 CBH2.L144W/T154V/R410S/S413Y CBH2.L144W/T154C/S413W 0.14 1.09 0.44 0.78 1.17 1.23 CBH2.R410S/S413Y 0.12 0.92 0.68 1.10 0.89 1.06 CBH2.L111S/L144Q/T154V/Y162N/R410S/S413Y 0.12 1.23 0.54 1.02 0.95 1.20 CBH2.L111S/T154C/R410S/S413W 0.39 0.64 0.54 0.64 1.15 1.13 CBH2.L144W/R410S/S413Y 0.27 0.99 0.63 0.74 1.03 1.39 CBH2.L144Q/T154C/Y162N/S413Y 0.09 1.72 0.54 1.35 1.01 1.21 CBH2.L144Q/Y162N/R410S/S413W 0.13 1.65 0.64 1.27 1.04 1.20 CBH2.L111S/L144W/T154C/Y162N/R410S/S413Y 0.11 1.15 0.46 0.91 1.16 1.15 CBH2.L144W/T154V/Y162N 0.19 0.95 0.80 1.00 0.94 0.72 CBH2.L111S/T154C/Y162N/R410S/S413Y 0.23 0.50 0.56 0.79 1.03 1.31 CBH2.L144Q/T154C/S413W 0.32 0.95 0.75 0.79 1.06 1.32 CBH2.L144W/T154V/S413Y 0.20 1.18 0.73 0.72 0.84 0.53 CBH2.T154V/Y162N/R410S/S413Y 0.65 0.69 0.78 0.96 1.15 1.51 CBH2.L111S 0.61 1.25 0.95 1.02 1.00 1.14 CBH2.T154V/R410S/S413W 0.17 1.28 0.44 0.75 1.06 1.28 CBH2.L111S/L144W/T154V/R410S/S413W 0.15 3.33 0.52 0.68 0.93 1.45 CBH2.T154C/Y162N/S413Y 0.15 1.01 0.70 1.12 1.09 1.16 CBH2.L111S/L144W/R410S/S413W 0.08 1.75 0.43 0.94 0.99 1.16 CBH2.L144W/T154C/Y162N/S413Y 0.12 2.38 0.67 1.43 0.92 0.96 CBH2.L144W/Y162N 0.16 0.80 0.83 1.13 0.93 0.72 CBH2.T154C/Y162N/R410S/S413W 0.25 1.88 0.59 0.96 1.10 1.43 CBH2.L111S/L144Q/T154C/R410S/S413Y 0.12 0.00 0.09 0.36 0.43 0.36 CBH2.L111S/L144W/Y162N/R410S CBH2.L144W/T154C 0.13 2.35 0.56 0.99 0.94 1.13 CBH2.L111S/L144W/T154C/S413Y 0.54 0.95 0.89 0.99 1.08 1.26 CBH2.L111S/L144W/Y162N/R410S/S413Y 0.20 1.01 0.57 0.88 1.12 1.31 CBH2.L111S/R410S/S413W 0.62 0.43 0.69 0.66 1.12 1.39 CBH2.L111S/L144Q/T154V/R410S/S413Y 0.50 0.74 0.68 0.75 1.15 1.45 CBH2.L111S/L144Q/R410S 0.61 1.00 0.76 0.83 0.99 1.03 CBH2.L111S/L144Q/T154C/S413Y 0.54 0.88 0.81 0.89 1.07 1.29 CBH2.L111S/L144Q/T154V/R410S CBH2.L111S/L144W/Y162N/S413Y 0.18 3.03 0.63 1.01 1.03 1.18 CBH2.L111S/L144Q/T154C/Y162N/R410S 0.13 4.55 0.55 0.79 0.65 0.75 CBH2.T154V/R410S/S413Y 0.51 0.88 0.71 0.82 1.10 1.38 CBH2.L144W/T154V/Y162N/R410S/S413Y 0.22 2.00 0.58 0.85 1.15 1.33 CBH2.L144W/T154V/Y162N/S413Y 0.02 4.80 0.12 0.13 1.26 1.41 CBH2.L111S/L144Q/R410S/S413W 0.45 0.42 0.66 0.85 1.12 1.30 CBH2.L111S/L144W/T154V/R410S/S413Y 0.70 0.58 0.75 0.82 1.10 1.43 CBH2.L111S/L144W/Y162N 0.13 2.62 0.64 0.58 0.92 0.80 CBH2.L144Q/T154C/Y162N/R410S/S413Y CBH2.L111S/T154V/S413Y 0.59 0.66 0.90 1.00 1.10 1.43 CBH2.L111S/T154V/Y162N/S413W 0.09 5.39 0.46 0.80 0.83 1.58 CBH2.L111S/Y162N/S413W 0.15 2.13 0.48 0.77 0.95 1.12 CBH2.L111S/L144W/T154C 0.57 1.70 0.94 0.97 0.92 1.02 CBH2.L111S/L144Q/Y162N/R410S 0.17 2.48 0.54 0.93 0.96 0.95 CBH2.L111S/T154V/Y162N 0.29 1.41 0.90 0.96 0.89 0.97 CBH2.L111S/L144W/S413Y 0.80 0.78 0.90 0.86 1.07 1.43 CBH2.T154V/Y162N CBH2.L111S/T154C/S413Y 0.65 0.89 0.89 1.19 1.16 1.36 CBH2.L111S/L144W/T154V/S413Y 0.60 0.98 0.90 0.93 1.11 1.32 CBH2.L111S/L144W/T154V/R410S 0.52 0.88 0.74 0.81 1.04 1.27 CBH2.L111S/L144W/T154C/S413W 0.42 0.81 0.76 0.78 1.10 1.39 CBH2.L111S/L144W/T154V/Y162N/R410S 0.24 1.65 0.65 0.95 0.95 1.09 CBH2.L111S/L144Q 0.74 1.29 0.95 0.95 0.93 0.92 CBH2.L111S/L144Q/T154V/S413Y 0.54 1.49 0.90 1.01 1.06 1.24 CBH2.L111S/L144Q/S413Y 0.77 1.20 0.92 0.89 1.01 1.17 CBH2.L144Q/R410S/S413Y 0.32 0.44 0.83 1.29 1.10 1.26 CBH2.L111F/L144W/T154V/Y162N/S413Y 0.12 0.88 0.59 1.30 0.96 1.19 CBH2.L111S/L144Q/T154C/Y162N/S413W 0.12 0.04 0.26 0.82 1.05 1.05 CBH2.S313T/S316P/G384Q 1.01 1.08 1.22 1.12 1.41 CBH2.P98L/S316P/G384Q 0.48 1.01 1.31 1.15 1.56 CBH2.P98L/S313T/G384Q/N443I 0.14 0.94 1.08 0.90 0.31 CBH2.K194E/S313T/S316P/G384C 0.27 0.90 0.83 1.11 1.60 CBH2.P98L/K194E/S313T/S316F/G384Q 0.12 1.25 1.77 1.03 1.36 CBH2.P98L/K194C/G384Q CBH2.K194C/S316P 0.23 1.12 1.14 0.96 1.16 CBH2.K194E 0.41 1.05 0.97 0.92 0.91 CBH2.P98L/K194C/S313T/G384Q/N443I CBH2.K194C/S313T/S316P/G384Q/N443I 0.08 1.08 1.37 0.53 0.50 CBH2.P98L/G384C 0.36 1.18 0.95 0.98 1.17 CBH2.K194C 0.27 1.16 1.00 0.76 0.80 CBH2.K194E/G384C 0.21 0.79 0.78 1.05 1.10 CBH2.P98L/K194E/S313T/S316P/G384C/N443I CBH2.P98L/S313T/S316P/G384Q 0.50 1.01 1.11 1.21 1.61 CBH2.P98L/K194E/S316P/G384C/N443I CBH2.P98L/K194E/S316P 0.27 1.15 1.14 1.11 1.39 CBH2.S316P/G384Q/N443I 0.16 0.89 0.72 0.66 0.42 CBH2.P98L/K194E/S316P/G384Q 0.15 1.14 1.56 1.05 1.37 CBH2.P98L/S313T/N443I 0.19 0.99 1.11 0.71 0.29 CBH2.K194E/G384C/N443I 0.05 0.63 0.91 0.10 0.73 CBH2.P98L/K194C/G384C/N443I CBH2.K194E/S313T 0.38 1.12 0.99 0.87 1.04 CBH2.P98L/K194E/G384C 0.08 1.14 1.75 0.87 1.21 CBH2.K194E/S313T/S316P/G384Q 0.47 0.98 1.37 1.14 1.55 CBH2.K194C/G384C/N443I CBH2.K194E/S313T/S316P/G384Q/N443I 0.05 0.78 0.78 0.07 0.32 CBH2.P98L/K194E/S313T/G384Q/N443I 0.05 0.83 1.08 0.50 0.53 CBH2.K194C/S313T/N443I 0.09 1.07 0.72 0.44 0.09 CBH2.P98L/K194C/S316P/N443I 0.07 1.12 0.99 0.61 0.85 CBH2.P98L/K194C/S313T/G384C/N443I CBH2.P98L/S316P/G384C 0.33 1.10 1.20 1.05 1.35 CBH2.P98L/K194C/S316P/G384Q/N443I CBH2.K194E/S313T/G384Q/N443I 0.08 0.83 1.07 0.20 0.34 CBH2.K194C/S313T/S316P/N443I 0.11 1.15 0.92 0.52 0.53 CBH2.P98L/K194E/N443I 0.08 1.37 1.40 0.25 0.48 CBH2.P98L/G384Q 0.54 0.99 1.14 1.12 1.36 CBH2.P98L/S313T/S316P/G384Q/N443I 0.19 0.99 1.23 1.04 1.03 CBH2.S316P/G384C/N443I 0.07 0.63 0.83 0.63 0.42 CBH2.K194E/N443I CBH2.P98L/K194E/S313T/N443I 0.08 1.17 1.46 0.56 0.44 CBH2.P98L/K194E/S316P/N443I 0.11 1.04 1.26 0.84 0.94 CBH2.G384Q/N443I 0.16 0.99 0.80 0.42 0.27 CBH2.P98L/K194E/S313T/S316P CBH2.K194C/S313T/G384Q/N443I 0.06 0.87 0.91 0.18 0.36 CBH2.P98L/G384Q/N443I 0.13 1.15 1.18 0.41 0.37 CBH2.G384C/N443I 0.06 0.75 0.72 0.23 0.44 CBH2.K194E/S316P/G384Q/N443I 0.10 0.88 1.30 0.75 0.65 CBH2.K194E/S316P/G384C/N443I 0.05 0.69 0.83 0.59 0.45 CBH2.P98L/K194C/N443I 0.07 0.90 1.19 0.42 0.40 CBH2.K194E/S316P/G384Q 0.33 1.06 1.40 1.07 1.37 CBH2.K194E/S313T/S316P/N443I 0.16 1.09 1.18 0.78 0.54 CBH2.K194C/S316P/N443I 0.09 0.99 1.11 0.60 0.40 CBH2.K194E/S313T/N443I 0.13 1.10 1.10 0.40 0.06 CBH2.K194E/S313T/G384Q 0.54 1.12 1.16 0.97 1.06 CBH2.K194C/S313T/G384Q 0.28 0.99 0.85 0.86 1.01 CBH2.P98L/S316P/G384C/N443I CBH2.P98L/K194C/S313T/G384C 0.06 1.14 1.49 0.92 1.17 CBH2.S316P 1.26 0.94 1.13 1.19 1.39 CBH2.P98L/K194C/G384Q/N443I CBH2.P98L/K194E/S313T/S316P/G384Q/N443I 0.07 0.94 1.21 1.03 1.03 CBH2.K194E/S313T/G384C/N443I 0.05 0.65 1.37 0.30 0.35 CBH2.K194E/S316P/N443I 0.17 1.02 1.07 0.74 0.49 CBH2.P98L/S313T/S316P/G384C 0.37 1.00 1.43 1.09 1.50 CBH2.S313T/S316P CBH2.P98L/K194E/S313T/S316P/G384Q 0.11 1.01 1.54 0.98 1.40 CBH2.K194C/G384Q/N443I 0.06 0.92 1.13 0.24 0.57 CBH2.P98L/K194C/S313T/S316P/G384Q 0.08 1.29 1.45 1.01 1.52 CBH2.P98L/K194C/S313T/G384Q 0.10 1.37 1.77 0.86 1.17 CBH2.P98L/K194E/S313T/S316P/N443I 0.14 1.02 1.44 1.04 1.15 CBH2.G384Q CBH2.P98L 0.68 1.03 1.29 1.08 1.27 CBH2.P98L/S313T/S316P/N443I 0.29 1.02 1.10 0.94 0.88 CBH2.K194C/S313T/S316P/G384Q 0.37 0.99 1.15 1.08 1.40 CBH2.P98L/K194E/S313T/G384Q 0.22 1.19 1.28 0.93 1.18 CBH2.P98L/K194C/S316P 0.19 0.98 1.09 1.04 1.42 CBH2.R153Q/N161W/Q422V 0.05 3.70 0.66 1.20 0.39 0.66 CBH2.N161W/P233D/Q422V 0.06 0.57 0.87 0.93 0.42 0.41 CBH2.R203H/Q422V/P444Q CBH2.R153Q/N161A/R203H/P444Q CBH2.R153Q/R203H/P444Q CBH2.N161W/R203H/P233D/Q422V/P444Q CBH2.R153Q/N161A/P444Q CBH2.N161W/R203H/P233D/P444Q CBH2.N161A/R203H/Q422V/P444Q CBH2.R153Q/N161A/R203H/P233D/Q422V/P444Q CBH2.R153Q/R203H/Q422V 0.16 1.07 0.75 0.70 0.45 0.42 CBH2.R153Q/P233D/P444Q CBH2.R153Q/N161A/R203H/P233D/P444Q CBH2.N161W/R203H/P233D/Q422V CBH2.N161W/R203H/P444Q CBH2.N161A/P233D/Q422V/P444Q CBH2.R153Q/R203H/P233D/Q422V/P444Q CBH2.N161A/Q422V/P444Q CBH2.R153Q/N161A/R203H/P233D CBH2.R153Q/N161W/R203H/P233D/Q422V CBH2.N161W/P233D/Q422V/P444Q CBH2.R203H/P444Q CBH2.N161A/Q422V 0.58 0.73 1.04 0.65 0.60 0.27 CBH2.R203H/P233D/P444Q CBH2.N161A/R203H/P444Q CBH2.N161A/R203H/P233D/Q422V 0.18 0.51 0.73 0.57 0.24 0.26 CBH2.N161W/P233D 0.21 0.69 0.86 1.02 0.87 0.72 CBH2.R153Q/N161W/R203H/Q422V/P444Q CBH2.R153Q/N161A/P233D 0.15 0.81 0.65 0.73 0.80 0.75 CBH2.R153Q/N161A/R203H/P233D/Q422V CBH2.N161W/R203H/P233D CBH2.P233D/P444Q CBH2.P233D 0.84 0.49 1.00 0.82 1.01 1.00 CBH2.R153Q/P233D/Q422V/P444Q CBH2.N161A/P233D/Q422V 0.28 1.19 0.87 0.48 0.37 0.23 CBH2.N161A 0.83 1.01 1.05 1.05 1.16 1.13 CBH2.N161W/P444Q CBH2.N161W/Q422F/P444Q CBH2.N161A/P233D 0.45 0.97 0.96 0.90 0.95 0.89 CBH2.Q422V/P444Q CBH2.R153Q/N161A/R203H/Q422V CBH2.R153Q/N161A/R203H/Q422V/P444Q CBH2.R153Q/N161A/P233D/Q422V 0.12 0.00 0.50 0.20 0.24 0.62 CBH2.Q422V 0.79 1.00 1.02 0.61 0.67 0.37 CBH2.R153Q/N161A/R203H 0.15 1.28 0.59 0.48 0.79 0.81 CBH2.R153Q/R203H/P233D/P444Q CBH2.R153Q/N161W/P233D/Q422V/P444Q CBH2.N161A/R203H/P233D/P444Q CBH2.N161A/R203H/P233D 0.23 0.57 0.79 0.82 0.86 0.68 CBH2.R203H/Q422V CBH2.R153Q/N161A/Q422V/P444Q CBH2.R153Q/R203H/P233D 0.10 1.11 0.52 0.87 0.81 0.88 CBH2.N161W/P233D/P444Q CBH2.R153Q/R203H/P233D/Q422V 0.13 0.65 0.42 0.35 0.40 0.38 CBH2.R203H/P233D/Q422V/P444Q CBH2.R153Q/N161W/R203H CBH2.N161A/P233D/P444Q CBH2.R203H/P233D 0.46 0.94 0.91 0.78 0.98 0.79 CBH2.P233D/Q422V/P444Q CBH2.R203H/P233D/Q422V CBH2.R203H 0.59 1.02 0.92 0.86 1.05 1.07 CBH2.N161A/P444Q CBH2.R153Q/N161W/R203H/P233D CBH2.N161A/R203H/Q422V 0.26 1.17 0.94 0.80 0.46 0.26 CBH2.P233D/Q422V 0.56 1.36 0.91 0.58 0.22 0.19 CBH2.R153Q/N161A/Q422V CBH2.P444Q CBH2.N161W/R203H 0.22 0.53 0.74 0.90 0.90 0.76 CBH2.N161W/Q422V 0.37 1.16 0.92 0.69 0.48 0.22 CBH2.R153Q/P233D CBH2.N161W/R203H/Q422V/P444Q CBH2.P98L/S413W 0.22 1.15 0.56 0.74 0.16 0.23 CBH2.L144W/S313T/S316P 1.30 0.70 0.96 0.99 1.40 1.12 CBH2.P98L/L144W/S316P/Q422V 0.51 0.67 0.97 0.94 0.75 0.82 CBH2.L111S/L144W/S316P 1.23 0.96 0.93 1.01 1.31 1.12 CBH2.P98L/L111S/S313T/S316P CBH2.L111S/S316P/S413W 0.99 1.08 0.88 0.85 1.51 1.11 CBH2.L111S/L144W/S313T/S316P/S413W/Q422V 0.66 1.31 0.89 0.92 1.31 1.04 CBH2.P98L/L144W/S313T/S316P 0.66 1.53 1.06 1.32 1.30 1.12 CBH2.L144W/S313T/Q422V 0.63 1.50 0.97 0.97 0.27 0.59 CBH2.L111S/S313T/Q422V 0.72 1.71 0.98 0.92 0.44 0.61 CBH2.P98L/L111S/L144W/S313T/S316P/S413W 0.40 1.80 1.03 1.58 1.38 1.01 CBH2.S313T/S316P/S413W/Q422V CBH2.P98L/L111S/S316P 0.08 6.48 1.20 5.02 1.39 1.09 CBH2.L111S/S313T CBH2.P98L/L111S/S316P/S413W 0.11 2.45 0.92 3.57 1.47 1.06 CBH2.L144W/S313T/S413W/Q422V 0.50 0.95 0.97 1.13 0.94 0.79 CBH2.L111S/L144W/S316P/Q422V 0.70 1.37 0.90 0.89 0.72 0.87 CBH2.L111S/L144W/S413W/Q422V CBH2.Q422V 0.55 1.20 1.10 0.97 0.28 0.54 CBH2.P98L/L111S/L144W/S313T/S413W/Q422V CBH2.P98L/L111S/L144W/S413W 0.21 0.42 0.55 0.40 0.26 0.25 CBH2.P98L/L111S/S313T 0.56 1.68 1.09 1.51 1.31 1.05 CBH2.P98L/L111S/L144W/S413W/Q422V 0.18 2.34 1.07 1.55 0.67 0.67 CBH2.L111S/L144W/S313T/S413W/Q422V 0.33 1.64 1.08 1.22 0.80 0.74 CBH2.S313T 1.18 0.49 0.97 0.94 1.13 1.07 CBH2.L111S/Q422V CBH2.P98L/L111S/L144W/S313T/Q422V CBH2.P98L/L111S/S313T/S413W 0.42 1.17 0.89 1.21 1.52 1.07 CBH2.S313T/S413W CBH2.P98L/S313T/S413W 0.35 0.80 0.92 1.25 1.37 1.07 CBH2.P98L/L111S/S413W/Q422V 0.06 6.43 0.93 3.08 0.82 0.77 CBH2.P98L/L111S/S313T/Q422V CBH2.S316P 0.92 1.17 1.02 1.15 1.34 1.12 CBH2.P98L/L144W 0.54 1.54 1.07 1.19 1.11 0.94 CBH2.L144W/S413W/Q422V 0.33 1.57 0.92 0.86 0.53 0.70 CBH2.L111S/L144W/S313T/S316P/S413W 0.54 1.63 1.05 1.39 1.18 1.02 CBH2.L111S 0.34 1.56 0.54 0.34 0.33 0.13 CBH2.P98L/L111S 0.69 1.36 1.04 1.38 1.30 1.03 CBH2.P98L/L111S/L144W 0.49 1.37 1.02 1.45 1.12 1.05 CBH2.P98L/L111S/S313T/S316P/Q422V 0.54 1.38 1.03 1.22 1.07 1.00 CBH2.P98L/S316P/S413W CBH2.P98L/S313T/S413W/Q422V 0.06 5.98 0.95 3.57 0.89 0.78 CBH2.P98L/L144W/S313T/Q422V 0.05 7.12 0.98 3.45 0.30 0.42 CBH2.P98L/S313T/S316P/Q422V 0.14 3.62 1.14 2.12 0.96 0.82 CBH2.L111S/L144W/S313T 0.76 0.78 0.98 1.06 1.01 0.96 CBH2.L111S/L144W/S316P/S413W/Q422V 0.81 0.82 0.79 0.77 0.93 0.85 CBH2.P98L/L111S/L144W/S316P/S413W/Q422V 0.38 1.47 0.95 1.01 0.50 0.41 CBH2.L111S/S313T/S316P/Q422V CBH2.P98L/L111S/Q422V CBH2.P98L/L111S/L144W/S313T/S316P/Q422V 0.07 3.16 1.06 3.78 0.75 0.75 CBH2.P98L/L111S/L144W/S313T 0.42 1.68 0.95 1.42 1.31 1.11 CBH2.L111S/S316P 0.56 2.08 0.90 1.21 1.23 1.01 CBH2.P98L/L111S/L144W/S313T/S316P 0.59 1.47 0.97 1.38 1.46 1.15 CBH2.L111S/L144W 0.22 0.96 0.53 0.35 0.16 0.10 CBH2.L111S/L144W/S413W 0.77 1.22 0.82 0.86 1.36 1.12 CBH2.L144W/S313T/S316P/S413W CBH2.P98L/L111S/L144W/S313T/S316P/S413W/Q422V 0.49 0.30 0.83 0.78 1.00 0.85 CBH2.P98L/L111S/L144W/S316P/S413W 0.49 1.26 0.97 1.13 1.29 0.97 CBH2.L111S/S313T/S413W/Q422V 0.27 2.43 1.01 1.84 0.96 0.85 CBH2.P98L/Q422V 0.56 1.65 1.06 1.05 0.59 0.79 CBH2.P98L/L144W/S313T/S413W 0.26 1.01 0.69 0.55 0.33 0.13 CBH2.L111S/L144W/Q422V 0.70 1.40 0.94 0.88 0.28 0.61 CBH2.S313T/Q422V 0.97 0.93 1.04 0.76 0.38 0.70 CBH2.P98L/S316P/Q422V 0.49 1.19 1.05 1.38 1.04 1.03 CBH2.S313T/S413W/Q422V 0.16 3.56 1.05 1.87 0.77 0.67 CBH2.P98L/L111S/L144W/S316P 0.43 1.36 0.83 0.52 0.12 0.00 CBH2.P98L/S316P 0.79 1.30 1.12 1.24 1.38 1.03 CBH2.P98L/L144W/S413W/Q422V 0.36 1.64 0.94 0.98 0.35 0.46 CBH2.L111S/L144W/S313T/S413W 0.40 1.30 0.91 0.51 0.86 0.74 CBH2.P98L/L111S/L144W/Q422V CBH2.P98L/L111S/S316P/Q422V CBH2.P98L/S413W/Q422V 0.32 1.50 1.06 1.55 1.14 0.93 CBH2.P98L/S313T/Q422V 0.27 0.65 0.88 0.23 0.08 0.08 CBH2.P98L 0.79 1.21 1.10 1.37 1.16 1.00 CBH2.L144W/S316P/S413W/Q422V 0.65 1.38 0.86 0.90 1.07 0.91 CBH2.L111S/L144W/S313T/S316P 1.00 0.91 0.98 1.11 1.36 1.09 CBH2.S413W 0.45 0.48 0.32 0.17 0.69 0.31 CBH2.L144W/S316P/S413W 0.55 1.44 1.05 1.18 1.20 1.00

Example 4

Classification of CBH2 Positions

As described throughout, functionality of cellulase variants was quantified as a performance index (PI), which is the ratio of performance of a variant to a parent or reference cellulase. Various terms set forth below are used to describe the mutation: up mutations have a PI>1; neutral mutations have a PI>0.5, non-deleterious mutations have a PI>0.05; deleterious mutations have a PI=0.05; combinable mutations are those mutations for which the variant has Performance index values=0.5 for at least one property, and >0.05 for all properties. Combinable mutations are mutations that can be combined to deliver proteins with appropriate performance indices for one or more desired properties. Positions at which mutations occur are classed as follows: Non-restrictive positions have ≧20% neutral mutations for at least one property; and Restrictive positions have <20% neutral mutations for activity and stability.

As shown in Table 4-1, for the 162 positions tested in CBH2, all 162 are Non-restrictive. Non-restrictive positions are the positions that are most suitable for use in constructing combinatorial libraries, since they have a large number of combinable mutations.

TABLE 4-1 Non-restrictive Positions of CBH2 PASC PCS Res. Res. HPLC PI Sp. Ac. Sp Ac. EtOH Heat # at % PI % PI % PI % PI % Position WT AA Position >0.50 >0.50 >0.50 >0.50 >0.50 5 S 19 47% 100% 84% 100% 100% 18 P 19 58% 84% 63% 95% 95% 19 T 17 71% 82% 76% 100% 100% 28 V 19 74% 89% 89% 100% 100% 30 S 19 53% 89% 63% 100% 100% 32 D 19 32% 100% 89% 95% 95% 35 S 15 67% 93% 100% 100% 100% 38 L 5 60% 80% 80% 100% 100% 63 R 19 84% 95% 95% 95% 100% 77 R 4 25% 100% 75% 100% 100% 79 P 14 50% 100% 100% 100% 100% 80 P 14 57% 100% 100% 100% 100% 89 S 19 58% 100% 100% 100% 100% 94 V 11 27% 91% 64% 91% 100% 98 P 19 42% 63% 47% 100% 100% 100 A 16 6% 75% 56% 81% 44% 102 A 19 58% 84% 84% 100% 95% 103 Y 18 72% 89% 67% 67% 67% 104 Y 19 11% 58% 11% 26% 21% 105 A 7 0% 71% 71% 57% 43% 107 E 12 58% 75% 33% 17% 0% 111 L 19 74% 95% 89% 95% 68% 117 T 18 83% 94% 100% 100% 94% 119 A 19 42% 100% 79% 100% 100% 120 M 19 32% 84% 47% 84% 74% 121 A 19 5% 63% 37% 63% 53% 125 A 19 37% 89% 26% 68% 63% 126 A 18 61% 83% 89% 89% 89% 129 K 8 13% 100% 88% 100% 75% 133 F 12 0% 17% 0% 67% 83% 134 M 19 79% 95% 89% 100% 100% 137 D 19 16% 37% 16% 100% 100% 138 T 19 74% 84% 74% 100% 100% 139 L 19 42% 89% 79% 100% 95% 140 D 19 21% 89% 74% 100% 100% 141 K 16 75% 75% 0% 100% 94% 143 P 19 63% 89% 89% 100% 100% 144 L 18 33% 94% 89% 100% 100% 147 Q 16 25% 94% 81% 100% 100% 150 A 19 47% 89% 79% 100% 100% 153 R 19 0% 53% 79% 95% 95% 154 T 19 26% 89% 89% 100% 95% 157 K 16 44% 100% 94% 100% 100% 158 N 18 33% 94% 83% 89% 94% 161 N 18 72% 100% 94% 94% 94% 162 Y 19 16% 84% 79% 100% 100% 177 A 19 37% 68% 42% 100% 100% 178 A 19 58% 32% 16% 100% 100% 179 L 18 50% 83% 72% 94% 100% 180 A 19 84% 26% 5% 89% 89% 181 S 17 82% 6% 0% 94% 100% 182 N 18 89% 89% 72% 100% 100% 185 Y 17 24% 35% 24% 59% 65% 186 S 19 63% 95% 79% 100% 100% 188 A 18 56% 83% 72% 100% 100% 189 D 18 11% 89% 72% 94% 100% 190 G 18 39% 83% 61% 89% 100% 191 G 14 0% 7% 7% 71% 93% 192 V 16 25% 75% 31% 81% 94% 193 A 18 6% 94% 89% 100% 100% 194 K 19 47% 74% 89% 89% 95% 196 K 9 44% 100% 78% 100% 100% 197 N 17 47% 100% 88% 100% 100% 201 T 19 5% 100% 79% 100% 100% 203 R 16 69% 94% 94% 100% 100% 204 Q 18 72% 94% 94% 94% 94% 206 V 15 33% 67% 40% 100% 100% 207 V 17 82% 82% 82% 100% 100% 210 S 14 21% 100% 93% 100% 100% 214 T 18 44% 83% 61% 94% 94% 225 N 19 0% 11% 5% 100% 58% 226 L 15 7% 33% 7% 93% 100% 228 T 15 7% 13% 33% 60% 80% 229 N 19 26% 79% 68% 100% 100% 230 L 19 16% 89% 26% 95% 74% 231 G 18 39% 67% 50% 100% 94% 232 T 14 21% 79% 93% 93% 100% 233 P 19 11% 100% 95% 100% 100% 234 K 13 54% 100% 31% 100% 100% 236 A 19 11% 89% 79% 95% 95% 237 N 16 75% 100% 44% 100% 100% 239 Q 18 89% 100% 89% 94% 100% 240 S 19 74% 89% 63% 100% 100% 243 L 19 53% 58% 47% 95% 95% 245 C 18 0% 22% 17% 78% 100% 247 N 18 83% 83% 39% 100% 100% 251 T 19 26% 89% 89% 100% 100% 252 Q 19 47% 95% 84% 100% 100% 254 N 17 6% 29% 24% 88% 100% 258 V 19 16% 37% 42% 89% 100% 266 H 16 0% 6% 0% 88% 94% 267 A 18 17% 33% 11% 78% 67% 268 G 19 95% 32% 21% 89% 89% 272 W 19 89% 58% 0% 63% 11% 274 A 19 11% 89% 42% 100% 84% 275 N 16 75% 100% 75% 94% 94% 281 Q 11 100% 100% 36% 100% 82% 285 N 14 21% 93% 43% 100% 100% 288 K 16 13% 75% 31% 100% 100% 289 N 18 6% 56% 50% 100% 100% 291 S 17 88% 82% 76% 100% 100% 292 S 19 63% 100% 74% 100% 100% 293 P 19 0% 11% 32% 79% 100% 294 R 19 79% 95% 95% 100% 100% 303 V 17 6% 18% 6% 71% 76% 304 A 19 79% 11% 5% 100% 95% 306 Y 17 6% 65% 6% 6% 29% 307 N 19 0% 21% 5% 32% 79% 312 T 18 94% 94% 50% 33% 17% 313 S 17 71% 100% 82% 94% 71% 316 S 19 84% 89% 84% 100% 95% 319 Q 18 89% 100% 72% 94% 94% 322 A 18 72% 94% 83% 94% 94% 323 V 17 41% 100% 35% 100% 100% 327 K 19 32% 95% 74% 95% 100% 328 L 19 68% 89% 37% 32% 26% 331 H 19 11% 63% 58% 68% 16% 338 A 18 67% 100% 100% 100% 100% 339 N 19 79% 95% 95% 100% 100% 340 H 19 21% 58% 68% 68% 37% 343 S 18 89% 89% 83% 100% 100% 344 N 18 39% 100% 56% 100% 100% 346 F 19 47% 84% 79% 100% 100% 356 K 19 79% 100% 63% 21% 26% 360 G 16 69% 75% 6% 13% 13% 361 Q 19 37% 74% 0% 26% 32% 362 Q 19 95% 95% 84% 95% 95% 363 Q 19 53% 95% 63% 89% 100% 364 W 18 89% 94% 6% 6% 6% 365 G 17 47% 94% 12% 100% 94% 371 I 19 26% 63% 58% 100% 79% 378 R 19 0% 47% 63% 32% 79% 380 S 19 95% 100% 26% 47% 37% 381 A 19 63% 89% 89% 95% 100% 382 N 15 80% 87% 0% 100% 100% 384 G 17 65% 88% 82% 100% 100% 386 S 17 88% 88% 76% 94% 100% 394 V 19 16% 42% 26% 58% 68% 396 P 19 5% 47% 68% 26% 47% 399 E 19 74% 5% 0% 26% 11% 400 C 19 63% 32% 11% 47% 32% 405 D 19 79% 95% 32% 74% 84% 406 S 19 84% 84% 84% 84% 79% 407 S 19 84% 84% 63% 74% 74% 410 R 19 84% 100% 74% 95% 100% 413 S 19 68% 100% 89% 100% 100% 414 H 19 63% 74% 63% 84% 63% 416 A 18 28% 78% 33% 50% 17% 417 L 17 59% 100% 47% 47% 18% 422 Q 18 72% 83% 28% 89% 94% 426 Q 19 74% 95% 63% 95% 74% 427 A 17 88% 41% 12% 76% 53% 429 A 19 58% 84% 74% 79% 68% 431 F 17 29% 82% 18% 18% 24% 433 A 19 63% 95% 74% 63% 47% 436 V 19 68% 84% 53% 37% 21% 440 T 19 74% 89% 63% 89% 89% 441 N 19 0% 74% 79% 37% 32% 443 N 16 38% 69% 44% 88% 94% 444 P 19 37% 63% 37% 95% 100% 445 S 16 31% 94% 63% 69% 56% 447 L 19 37% 89% 89% 100% 68%

Example 5 Effect of Charge Change on the Activity of CBH2 Variants

In this example, the effect of charge change on the activity of CBH2 in PCS and PASC assays was assessed. Briefly, the number of PCS and PASC winners in the CBH2 SELs was determined as a property of net charge change. In Tables 5-1 and 5-2, the ratio of observed to expected (o/e) winners was determined in PCS and PASC assays respectively. Values in bold italics are significantly different from the average of 10 random distributions plus or minus the number of standard deviations (sd) listed in the respective columns

TABLE 5-1 Charge Effect on Activity of CBH2 Variants in a PCS Assay PCS Charge change o/e 1 sd o/e 2 sd o/e 3 sd Results −2.00

1.20 1.20 >90% confident more than expected −1.00

>99% confident more than expected 0.00 1.03 1.03 1.03 as expected 1.00

0.46 >95% confident less than expected 2.00 0.00 0.00 0.00 as expected

TABLE 5-2 Charge Effect on Activity of CBH2 Variants in a PASC Assay PASC Charge change o/e 1 sd o/e 2 sd o/e 3 sd Results −2.00

>99% confident less than expected −1.00

0.82 0.82 >90% confident less than expected 0.00

1.06 >95% confident more than expected 1.00 1.03 1.03 1.03 as expected 2.00

0.40 >95% confident less than expected

As shown in Table 5-1 and FIG. 2A, decreasing charge (e.g., −1, −2) results in a significantly higher frequency of CBH2 winners in the PCS assay, while increasing charge (e.g., +1) results in a significantly lower frequency of CBH2 winners in the PCS assay. In contrast, as shown in Table 5-2 and FIG. 2B, a significantly higher frequency of CBH2 winners is observed for variants without a charge change (e.g., 0), while a significantly lower frequency of CBH2 winners is observed for charge changes (e.g., −2, −1, +2).

In conclusion, CBH2 activity on PCS correlates with decrease in charge. CBH2 activity on PASC however, shows that this trend does not correlate with cellulose hydrolyzing activity, but is specific for PCS material.

Example 6 Creation of Trichoderma reesei Screening Strains

Improved screening strains were created to increase the consistency of CBH2 variant expression in the presence of factors unrelated to the amino acid sequences of the enzyme variants. In particular, T. reesei screening strains were developed in combination with a targeting vector to force integration of cbh2 variant genes (e.g., coding region in operable combination with a regulatory sequence). The new strains prepared during development of the present disclosure, combine several mutations that are advantageous for screening variant libraries. A schematic of the genetic engineering steps is shown in FIG. 4.

Deletion of ku80 from the T. reesei quad deleted derivative strain. A single orthologue of MUS52, the N. crassa orthologue of the human KU80, was identified by TBLASTN search in the genome sequence of H. jecorina QM6a (Trichoderma reesei) and was consequently named T. reesei ku80. protein id 58213; http://genome.jgi-psf.org/Trire2/Trire2.home.html The nucleotide sequence of the T. reesei ku80 gene is provided as SEQ ID NO:23:

ATGGCGGACAAGGAAGCAACCGTCTTCATCATCGACCTCGGCGCGTCCA TGGCAGCTGTCAATGGGGGTCGAGAAGAATCCGACCTTGATTGGAGCAT GAGCTACGTCTGGGACAAGATCAGCAACGTCGTGGCCTCGAATCGCAAG ACGCTGTGCGTTGGCGTCGTGGGGTTCAGAACCGACGAGACAAACCACA CGCTGAGCGAGGATGGGTACGAGAACATCTCCATATTGCAGCCCCTGGG GCCGATGAGCATGTCCAGCCTCAAGGCTCTTCAGCCCAAGGTGAAGCCG AGCAGGACGGTGGAAGGCGATGCCATCTCGGCGATTGTCATTGCCGTCG ACATGATTGACAAGTACACGAAGAAGAACAAATGGAAGCGGCAGATTGT TCTCATTACCGACGGCCAAGGCGAGATTGATCCAGATGATATTGGCGAC ATTGCTAGAAAGATGCGCGACTCGAATATTGAATTGACAGTCTTGTGAG TTGGCGAGACCGTTTGGCGGACGGTAATGGTGCTGACGGTGATGCAAGG GGCGTCGACTTTGATGCTCCCGATTACGGCTTCAAAGAGGAGGACAAAC CTTCAGTCAAGGTACTCCATATGTTCACTTCTTTTCTTTTTCTTCTTTA TTTTCTTTTCTTTTGAAGCTTTCATTAACCTCTTCGTTAGAAGCAAAAC GAAGAGACCCTAAAAAAGCTCGTGGATGGCTGTGGCGACGACTCAAGGT TCGCCTCCATGGTCGAGGCCATTGACGACTTGAATGAGCCACGAGCAAA GTCGGTCAAGCCTTACAAAACGTACGAAGGTCTCTTGACCTTGGGAGAT CCGAAAAACGCTCCCGCAGTGGTGGAAATCCGCGTCGAGAGATACTTCA AGACCCATCTAGCCAGGCCACCTGCCGCCAGCACCGTGGTGGTCAAGGA GGAGCAAGCTGGGCCGTCTCAGGCAGACGAGGACGAACAGATGGACGGA GCGGAACTTACAGCTGTGAGGCAGGCCAGGACATACAAGGTCAATGATC CAGATGCCCCTGGCGGTAAGCGTGACGTTGAGTTTGAGTCTCTGGCCAA AGGGTACGAGTACGGCAGGACGGCAGTCCACATCAGCGAGTCTGATCAA AACGTCACCAAGCTCGCGACAGAAAAGAGCTTCAAGATCATCGGCTTCG TCCAGAAAGAAAAGGTATTGGCTTGGCTCTCAGCATTTGACCCGTTGCT CTTGGCTAACCCTTGTTTAGTATGAAATGCTCCTTAATCTTGGCGAAAC CTGCGTTACCGTTGCATCCAAGTACGATGAAAAGTCTGAGCTGGCTTTT AGCTCTCTGGTGTGGGCGCTCTCGGAGCTCGACGCCTACGCCGTGGCCC GCCTAGTAACTAAGGACCAAAAGGACCCCATGCTGGTGTTACTGATGCC GTATATGGAGCCTGATTATGTTTGTCTCTATGATGTGCCTCTGCCTTTC GCAGAGGACATCAGGACGTACCAGTTTCCTCCCTTGGACAGAGTCGTTA CCGTCAGTGGCCAAACGCTCACCAACCATCGCCTATTGCCATCCGACGA GCTCAACCAAGCGATGAGCGACTACGTAGATGCCATGGACATTTCAAGT TATGGTATCGATGAAGATGGGTGAGTATAGAAGATGATTGTTCAAATCT TTCACTTCTAAGCATTGCTTCTGATCTAGGCAACCGGCTGAATATGCCA CCATCGATGAGTTATACAACCCTGCGATACATCGCATAGGCCATGCGAT CAAACAACGAGCGATCCACCCAGAGAAACCCGTGCCCGAGATCCCCCCA GTCTTGCTTAGATTCGCAGCACCCCCGACAGAACTCGTCGAGACTGTGC AGCCTCATATCGATGCACTGATTCACGCTGCAGACGTGAAGAAAGGTAC TGATTCCATTACATATGCTTCTCTGCACACTGATGTTTGATTTGTGCTA ACGCCCCCCTTAGTGCCGCCCAAGGCCAAGGGCAAGCGCCAAAGAGAAA CAGTTAAACCCATCTCGGGACTGGATGTGGATGCCCTTCTGGGAGAAGA GCAGAAAGGTTCCATTAGTCCGGAGAATGCCATTCCGGACTTCAAACGA GCCCTCAACTCGTCCGAAGAAGTCGAGCAGATTGCCGACGCCACAAAAC AAATGGGGGCCATTGTGCGGTCTCTCATTACGGACAGCTTCGGGGATAG CAAATATGCCCAGGCAATGGAAGGCATTGGTGCGATGCGTGAGGAGCTG ATCAACCTGGAAGAGCCTGGCCTGTACAACGACTTTGTGCGCGACTTGA AGAAAAGTTTGCTATCTGGAGCCTTGGGTGGTGACAGGCGAGATTTCTG GTTCAAGATGAGGTGGGCGAAGCTGGGCCTGATTGACAAGAAACAGTCG GAGGTGTCTTCGGTCACTCTTGAGGAGGCGGACGAGGTGAGTGGTGCAG CATGCTGTCGGATTATACGGACGTTGTTTGCTAACTTGTGGGATAGTTT TACAAGTCGAGGTGAGGTATCTACGTTGACCAAGAATGGGACCATGTAT ATGAGCGGTGTAACAACAGAATCCTGTGCTTTGAGCATTGTATGA

The T. reesei ku80 gene was deleted from the quad deleted derivative strain using standard methods of the art (WO 2005/001036). Briefly, a ku80 deletion cassette was utilized that employed a selectable marker flanked between 1.3 kb of 5′ ku80 sequence and 2.3 kb of 3′ ku80 sequence, as schematically shown in FIG. 5. The variant T. reesei als, which confers resistance to the herbicide chlorimuron ethyl, was used as selectable marker as described in WO 2008/039370. The nucleotide sequence of the ku80 knockout cassette is 7685 base pairs in length: bases 1-1271 correspond to the 5′ ku80 homologous region; bases 1280-7685 correspond to the als-chlorimuron ethyl resistant variant (A190D); and bases 5381-7685 correspond to the 3′ ku80 homologous region. The nucleotide sequence of the ku80 knockout cassette is provided as SEQ ID NO:14:

ggccgcctcaacacccacactcgaggcacacgagttcatcggcggcaccc ccacaagctctcggccaacctgctaccggctctctcgcgagacttcccaa agcctacaaacgaggtcgacgtcaaggaggccctcgagcgccagcccggc agatggagcctccagggccagatcaaggccaacaacatgagagcccagag cgccgcactccggctcgacgacaaggagggcaaggcgagagcctttgagg aggccaagcgcgagctactggcgtatcaccacagcgccctgcggaagcct tccggcgcaagataatgagcttgatcgcaatgacgagttcacgtacgctt tgccatattgttgttgctttttgtttggtcctacatgtacggcgcattgg ttgggaggatatacccacggagagtgtccgagtggcttctgggatttaga gcgtcattagcaggatagagatggttggccaggggaatggaattgacttt tcactacaaggaacttgttcactctggtgttgattcccattgcgtgactg gtagtagggaggaatgcttttactttgtgccactagaccgcagagaaggg ttggttgcaagcggggtccgtgtataccgaccaagagtgatgggcataca gcaacgtttctgaacgacttcattttgtccgagtctactggatgcgagat gccagcgtgaagccgtacgccaccagggcgacgaactcgacaaggttgac gagggaggagatgccgtgcagcatgccaaacacttgttgagggcacgcat ctcatccgactgtgcatccttgtcataccactcctttccgtctcgcttgg ctggtgggagggacaacaaatccatcgtcagccatccggggtctcaaatc aatggcgtgcatgcggagtcgggcttgaggctaaccttgtccatggcggt ccttcatggtcttgacagtggcgggaagcagcacggcgaggttgacgagg ccgctgacgaacatggttgcgatgggcaccaaggagctccacttgttggg agcgtcgacgaggccgccgatgccgcccttgatgcccaagagggcgtttc cggggaacgtgagggcgagcagcgcggggatggccgtctgcatgccaaag tagatggggaacagcttgctctggatggcggagaaggagggccggctgac ggtgcggaacatgacgatgccgttgacgaaggactgcagtagcgtagtgt gatggtaagcagctggccggcgcgcctgagacaatggccggcaatggtaa aaaggaccaagatgtactaggtagttgcaatgtggcttattacctaccta ctacctggtaggcacctactaggtacttgggtagacggacaatgaaattt gaagtcggggttgcaggaaagcagggcgctggacacattgtgcttcaggc ggtacccgtcgtcatcgtcagccaatgtcgaggcccggcagcccgaggag cgagacaaccttggccggaggagcccgcaggtacctgccaaagcgcggct ggtacctctcaaccctctcaggcctgttggatgccctatgacatgccctg ggggatgcagctgttgccccggccccgcactttcgggtgaccgcgaggct gctgattggctggttgccacgggctgggcggtccctgaagttgttgccat ctgaactctgtcggcgctggcgtcggctgcgcccaatgggaggcgagaca actcagggtactagaatcactgacagaagaagagaatcgaaagtaggtag acagccaattcgttgcatggcaggcaaccgcacaggagaaaaattgacta ccccacaatcaggcacagtaagtagggcacagtacgtatgtacagacaag gcgcaagcgatactgcgcgacccggtacctcgccggcttgacacgtgcga caggctactttactagtattcgcagcggcgggtcgcgcattattacatgt actgtgccgccatttgatgactgggctgctgcagtattagtagatctgcc cggcatcgcccttccatgggcgcgacccgggactggaccctctgactcta cctacatgtacctaggccgggccgggcttggtgacttttgtccgatcagg tcgttcgcctggctacctattatttctctttcttcttctccatcctgctt ctggccttgcaattcttcttcgccactcctccctcttccccccgcgatac ccttgaattcgtcagagaggaaaagacgagaaaaaaaagggcagcagaga cgtcggtctggctcacgtgctgcatctctgcgcactctcattttttttat tgtccgacccctccctcaaccttctccttcgttgacaggctaagccttgc ttcgacgctctctctttgaatttttctacttctaccttcttttcttgcgt gttacccaccatagctcgattcacgatgctccgaagtcgccaagtcacag ccagggccgtccgggctctgggccaggcgcgcgcctttacctcgacgacc aagcctgtcatgatccagagcagccagaggaaacaggccaacgccagcgc tgctccgtaagtcgcccattgccattgcatcttctgtttgatatatactt cctgctgcttgcgtggcgtcgtctctcggttatgcgtgtcaaggaccagg tgtgttcgcatcgtggttttccagcgccgattaccgggggacgaattttt ggctgctcaactcgcgcgcgcgcattctgattcttcgttttcaatcttga gcgacaactggctaacataatggccattggcaattgcttcacacagacaa gtccgccctgtaccgagccctgctttcaacgctgaagacaaagaccgcag ccatgtgcagcctctggtcaacccgtcgaagcccgacatggatgaatcgt atgtccacgtcccctcgtcccgccctacaaaatgaacacgattacaccag aatttttgcaacaatcgacacttctataacagaccaattgagctttgttc tgaccaatcatgttgctctagattcattggcaaaaccggaggcgaaatct tccacgagatgatgctgcgacagggtgtcaagcacatttgtaggttccga tgccggccgcccacacgggctccatccttgctccatctctccagctaggc aaatctcgctaaccttgagtcaccatccagtcggataccctggcggcgct atcctgcccgtcttcgacgccatctacaactcaaaacacttcgacttcat cctgccccgtcatgagcagggagctggccatatggccgagggctatgccc gtgcctcgggcaaacccggtgtcgtcctggtgacttccggccccggtgct accaatgtcatcacgcccatgcaggatgccctgtcggacggaacgccctt ggtcgtcttctgcggccaggtccccaccacggccatcggcagcgatgact tccaagaggccgacgtcgtgggcatctcgcgggcctgcaccaagtggaac gtcatggtcaagagcgttgctgagctgccgcggagaatcaacgaggcctt tgagattgccaccagcggccgccctggccccgtcctcgtcgacctgccca aggatgtcacggctggtatcctgaggagagccatccctacggagactgct ctgccgtctctgcccagtgccgcctcccgcgccgccatggagctgagctc caagcagctcaacgcctccatcaagcgtgccgccgacctcatcaacatcg ccaagaagcccgtcatctacgccggtcagggtgtcatccagtccgagggc ggcgttgagctcctgaagcagctggcggacaaggcctccatccccgtcac caccaccctccatggcctgggtgcctttgatgagctggacgagaagtcgc tgcacatgctgggcatgcacggctcggcgtatgccaacatggccatgcag caggccgacctcatcatcgccctcggcagccgattcgacgaccgtgttac tctgaatgtctccaaatttgcgcctgcagccaggcaagctgctgccgagg gccgcggcggcatcattcactttgagatcatgcccaagaacatcaacaag gtcatccaggcgaccgaggccgtcgagggcgacgtcgccaccaacctgaa gcacctcattccccagattgccgaaaagtccatggcggaccgaggagagt ggttcggcctcatcaatgagtggaagaagaagtggcccctgtcaaactac cagcgcgcggagcgggctggcctcatcaagccgcagacggtcatggagga gattagcaacctgacggccaaccgaaaggacaagacgtacattgccacgg gtgtcggccagcaccagatgtgggttgcccagcacttccgctggaggcac cctcgatccatgattacctctggtggtctgggcaccatgggctacggtct ccccgcggccattggcgccaaggtggcccagcccgacgctctcgtaattg acgttgatggcgatgcctcgtttaacatgacgctgacggagctgtcgact gctgcacagttcaacattggcgtcaaggtggttgtgctcaacaacgagga gcagggcatggtgacgcagtggcagaacctcttttacgaggaccgatatg cccacacgcaccagaagaaccccgacttcatgaagctggccgacgccatg ggcgttcagcaccagcgcgtgacggagccggagaagctggtcgatgccct gacgtggctgatcaacaccgatggcccggccctgttggaggttgtcacgg acaagaaggtgcctgtcctgcccatggtgcccgccggatcggccctgcac gagttcctcgtctttgaacctggtgagtctacttcagacatattgcttgc gcattgcagatactaacactctcacagaaaaggataagcagcgccgtgag ctgatgaaggagagaacaaagggtgtgcactcctaaagcgatgatgtctg cgaggggttcttcgttgaaccctagttcaggcaccatcttaccctcttat tttttcccgtgggctttcattttgtgtcatccgagcatgacgttgtaggg ttggagtttcttcctttttatcttgtcatttactggtacccataggcgcg agactaggcttccatgttttgttttgcgactttcaaaaagtacttttagt ggtttggggcacgacgagggggggcaacctcttctgtcgaaaaaggtggc tggatggatgagatgagatgagatgagggtgaagatagatacctgcagtg tttttgacgcgacgggatggcgatcgcagcacccccgacagaactcgtcg agactgtgcagcctcatatcgatgcactgattcacgctgcagacgtgaag aaaggtactgattccattacatatgcttctctgcacactgatgtttgatt tgtgctaacgccccccttagtgccgcccaaggccaagggcaagcgccaaa gagaaacagttaaacccatctcgggactggatgtggatgcccttctggga gaagagcagaaaggttccattagtccggagaatgccattccggacttcaa acgagccctcaactcgtccgaagaagtcgagcagattgccgacgccacaa aacaaatgggggccattgtgcggtctctcattacggacagcttcggggat agcaaatatgcccaggcaatggaaggcattggtgcgatgcgtgaggagct gatcaacctggaagagcctggcctgtacaacgactttgtgcgcgacttga agaaaagtttgctatctggagccttgggtggtgacaggcgagatttctgg ttcaagatgaggtgggcgaagctgggcctgattgacaagaaacagtcgga ggtgtcttcggtcactcttgaggaggcggacgaggtgagtggtgcagcat gctgtcggattatacggacgttgtttgctaacttgtgggatagttttaca agtcgaggtgaggtatctacgttgaccaagaatgggaccatgtatatgag cggtgtaacaacagaatcctgtgctttgagcattgtatgatatgattatt gatgaaccggacaaaagggggtaggggattgatgccatcacgaccgattg accagacctggattctcgcacagcatggctgctgattttgttgaccttgc gacgtaacatccctgaagaacaacctactattaacctatcatttagcaga agctctgtaaccttcttgattcttgtattcagcttctgagtctgtcaaat gtaatcatttcgaggttgtgtaattccggccaagcaggcggccgtctgcc agcgcctgcctaggctgcaccgcaatctgcccaatcagctgcccttcagt ttcgtttgaccttgcagctgcccttcatcctttatctgcacacaattatt ttcctctgctctgcgcattcttctctctctcgtctcccttctcaagctca acttcacctcatccgctccactacaagccctcccgtcgtcgtctcgcatc ctcatctcgactgcggccagcaaaacaagcaaagccgtgatcgatcctca gcatggctaccttcaacctcaccgtccgcctggagatgctcaaagaaatt ggaatcaccgtccaatacggcgagcatgtagcgaaagaagcagccagcaa cgaagcagcgatggcattcgaagaagaagaagagttccccgccgttgtgc cgcccaaggcagaacagcacgcctctgaacacgacgctggccacgatgct tgggacgcggctgcccacatctcgacttcggcgcaagaacagcagaagcc ccaggagatggacgactcgtctatcgtgatgccgctggactactccaagt ttgtcgttggagagcctgcggacgaatccatcagcttttgctcgtggaag gtcgtcgaggcttatcctgaccagtttatcggcaaggcaaacaggcctcg tgtatgtagcgattgctttctctgcattatgggaatctcaagagagtatg gtagaagataactgacaacttgcaggccaagccgtactttgacaagattt tggaagacagagtctgggatttgtgaggatcttgattgatgtgcatatgg cgacatgcctgctaatatcattgtagcttctatctctacaaccccgagaa gcatcagagaagcctcgcgtgctggtgcccactgttcagctcgaaggctt tctcaaaagcatcaacagagcgctcggtacttctctcaccattccaggag gggcaaaccaggaccgtttttatctgaggttcggccagggagacacccca aggcctcgatatctacagaggtcgagagaccagaaatccctaaagattga aacgttccccgattttcaacaggcggactacgacagctttaggaacgcgc atggcgccatccaggaggactggttgaagaactggcagatgctggtacct cggccgagtttcgacaagaagaaaaatgcagacaaaagagcagccaagag aaggctcgagcgagagcgaatgcttcacaatacgcaggaatttcttcatt tggcaggtaagggcaaaggggctgacgtgg.

Creation of the Archy2 strain from the T. reesei Δku80 quad deleted derivative strain. The pyr2 gene was deleted from the ku80 knockout strain. The pyr2 deletion cassette contains the T. reesei cbh1 promoter, a hygromycin resistance gene and a partial amdS selectable marker flanked by 5′ and 3′ pyr2 sequences, schematically shown in FIG. 6. Use of this vector permits screening for resistance to hygromycin and fluoroorotic acid of pyr2 knockout transformants. The partial amdS gene contains the 3′ portion of the gene, but lacks a promoter and the amino-terminal portion of the coding region, and is consequently non-functional. The nucleotide sequence of the pyr2 knockout cassette is 9259 base pairs in length: bases 1-1994 correspond to the pyr2 3′ homologous region; bases 2002-3497 correspond to the T. reesei cbh1 promoter; bases 3563-5449 correspond to the hygromycin resistance selectable marker; bases 5450-7440 correspond to the A. nidulans amdS 3′ partial marker; and bases 7441-9259 correspond to the pyr2 5′ homologous region. The nucleotide sequence of the pyr2 knockout cassette is provided as SEQ ID NO:15:

atcacgccctcgcataaaagaccctcaagagtccatgtgccctatctgcc tgatcttcctaacccttatttaacattggccctatcacaacctagttctt cttcagcctgctttgtcaacacttgtcacggttcaactcaacgtaatcag caggtagcaggacgaggatagggagagaaacgaagagaagaagaggagag aggaagaaaaaaaaaagaaaagaaagaaaaagggaaaaggaaagaaggag gaaaagagaagaaagtcagatgaagaagcaagaagacgccatggtagcca ccgcttgtcagggctccttagcaacgaacaactctagcttggggacttgt cgatgtgtcgtttccttcctacccatcagcaccaacgatgagttcgatat agacgaggacctcatggaagtagagaccattgggttcgacaggatctctc agtttcacttctatgaggtctgtcgctcggatgactttttgaggagcttc cccttctgcttcaaccccaaactctattcctgaaaccgcagcacgttggc acggccgtgttgctggagcagtttgctttcgagcactctcagcgtggttt cagcagcccactggtgagtggcctcctttgacgtccacaccttgctcctg tcgcatgcgtatctggtgggaacgactgctccaaggaggattgctaacga ggttgtaggccgaatatcgcatcagattctccggtaaccttagctacggc ctcttcaacatctgtgacatgacggagcgcaagtactggtggttggcgac caagatgcgcggctggaacatcgacggctgccccgaagacgtcaggagac tcatgtttgttcacatcatcgccaccctgggatgcagccccgtcgtgacg gatgaagacatggactaccccaagaactgggcggcaattctccacggtag agacagatatccgagtgaacctgtgggccaccggcctcatgggcgcacca tctgcctccactcggtggccgtctgccctcgtctccagggcttgggtctc ggtactgcgactctgaagtcgtatgtgcagcgcatgaacagcctcggcgc cgcggaccgtgttgctctcgtttgccgcaagcccgagacgagattttttg aaagatgcggcttcaggaacagcggccggagtagtatcaagactctggtc ggcgaatactacaacatggtgtgtgatccacatcgacttggccagactct atacgattttcaaacctcgctatacgtcatattgacttgtttctttaggt atcgatttgcccgggcccaaagactttatcgactggaatagcattgccga cgctgccaagaagatgtgaaccatttgactgatacgatgtgtgctacgca tgtcgaccttctttgtttgtttctttggcggctctttgtataccttggga cacggcagacgcatgtctatgtgaagaaaacgttcacggcgctgtttgca tcaggaatatgatcattaaacatggagcgtaatggtattaatgatcaact agaaaaatggtatggaagggcgagagggcgatcaacaaagcagcccgggg catagtctggaagcagcaggaattggaagggaaaaggaagctgcacaatg aagggatatcgtgagcggagtggctcacgagagtatcaacagactggcga aagcaagcaattgccaacgccggctattaggccataagatggcctgttgt gagtcccagttgcacgtatccccatatgactgctctgtcgctgacttgaa aaaaaatagggaggataaaggagaaagaaagtgagacaacccgtgaggga cttggggtagtaggagaacacatgggcaaccgggcaatacacgcgatgtg agacgagttcaacggcgaatggaaaatcttgaaaaacaaaataaaataac tgccctccatacgggtatcaaattcaagcagttgtacggaggctagctag agttgtgaagtcggtaatcccgctgtatagtaatacgagtcgcatctaaa tactccgaagctgctgcgaacccggagaatcgagatgtgctggaaagctt ctagcgagcggctaaattagcatgaaaggctatgagaaattctggagacg gcttgagaatcatggcgttccattcttcgacaagcaaagcgttccgtcgc agtagcaggcactcattcccgaaaaaactcggagattcctaagtagcgat ggaaccggaataatataataggcaatacattgagttgcctcgacggttgc aatgcaggggtactgagcttggacataactgttccgtaccccacctcttc tcaacctttggcgtttccctgattcagcgtacccgtacaagtcgtaatca ctattaacccagactgaccggacgtgttttgcccttcatttggagaaata atgtcattgcgatgtgtaatttgcctgcttgaccgactggggctgttcga agcccgaatgtaggattgttatccgaactctgctcgtagaggcatgagtg aatctgtgtcgggcaggacacgcctcgaaggttcacggcaagggaaacca ccgatagcagtgtctagtagcaacctgtaaagccgcaatgcagcatcact ggaaaatacaaaccaatggctaaaagtacataagttaatgcctaaagaag tcatataccagcggctaataattgtacaatcaagtggctaaacgtaccgt aatttgccaacggcttgtggggttgcagaagcaacggcaaagccccactt ccccacgtttgtttcttcactcagtccaatctcagctggtgatcccccaa ttgggtcgcttgtttgttccggtgaagtgaaagaagacagaggtaagaat gtctgactcggagcgttttgcatacaaccaagggcagtgatggaagacag tgaaatgttgacattcaaggagtatttagccagggatgcttgagtgtatc gtgtaaggaggtttgtctgccgatacgacgaatactgtatagtcacttct gatgaagtggtccatattgaaatgtaaagtcggcactgaacaggcaaaag attgagttgaaactgcctaagatctcgggccctcgggccttcggcctttg ggtgtacatgtttgtgctccgggcaaatgcaaagtgtggtaggatcgaac acactgctgcctttaccaagcagctgagggtatgtgataggcaaatgttc aggggccactgcatggtttcgaatagaaagagaagcttagccaagaacaa tagccgataaagatagcctcattaaacggaatgagctagtaggcaaagtc agcgaatgtgtatatataaaggttcgaggtccgtgcctccctcatgctct ccccatctactcatcaactcagatcctccaggagacttgtacaccatctt ttgaggcacagaaacccaatagtcaaccgcggactgcgcatcatgtatcg gaagttggccgtcatctcggccttcttggccacacctcgtgctagactag gcgcgccaggaagcccggaaggtaagtggattcttcgccgtggctggagc aaccggtggattccagcgtctccgacttggactgagcaattcagcgtcac ggattcacgatagacagctcagaccgctccacggctggcggcattattgg ttaacccggaaactcagtctccttggccccgtcccgaagggacccgactt accaggctgggaaagccagggatagaatacactgtacgggcttcgtacgg gaggttcggcgtagggttgttcccaagttttacacaccccccaagacagc tagcgcacgaaagacgcggagggtttggtgaaaaaagggcgaaaattaag cgggagacgtatttaggtgctagggccggtttcctccccatttttcttcg gttccctttctctcctggaagactttctctctctctcttcttctcttctt ccatcctcagtccatcttcctttcccatcatccatctcctcacctccatc tcaactccatcacatcacaatcgatatgaaaaagcctgaactcaccgcga cgtctgtcgagaagtttctgatcgaaaagttcgttcagcgtctccgacct gatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtag gagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctac aaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattcc ggaagtgcttgacattggggaattcagcgagagcctgacctattgcatct cccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactg cccgctgttctgcagccggtcgcggaggccatggatgcgatcgctgcggc cgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcg gtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccat gtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgc gcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtcc ggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaat ggccgcataacagcggtcattgactggagcgaggcgatgttcggggattc ccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgta tggagcagcagacgcgctacttcgagcggaggcatccggagcttgcagga tcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactcta tcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtc gatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaa atcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtact cgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaat agagtagatgccgaccgggatccacttaacgttactgaaatcatcaaaca gcttgacgaatctggatataagatcgttggtgtcgatgtcagctccggag ttgagacaaatggtgttcaggatctcgataagatacgttcatttgtccaa gcagcaaagagtgccttctagtgatttaatagctccatgtcaacaagaat aaaacgcgtttcgggtttacctcttccagatacagctcatctgcaatgca ttaatgcattggacctcgcaaccctagtacgcccttcaggctccggcgaa gcagaagaatagcttagcagagtctattttcattttcgggagactagcat tctgtaaacgggcagcaatcgcccagcagttagtagggtcccctctacct ctcagggagatgtaacaacgccaccttatgggactatcaagctgacgctg gcttctgtcagacaaactgcgcccacgagttcttccctgacgccgctctc gcgcaggcaagggaactcgatgaatactacgcaaagcacaagagacccgt tggtccactccatggcctccccatctctctcaaagaccagcttcgagtca aggtacaccgttgcccctaagtcgttagatgtccctttttgtcagctaac atatgccaccagggctacgaaacatcaatgggctacatctcatggctaaa caagtacgacgaaggggactcggttctgacaaccatgctccgcaaagccg gtgccgtcttctacgtcaagacctctgtcccgcagaccctgatggtctgc gagacagtcaacaacatcatcgggcgcaccgtcaacccacgcaacaagaa ctggtcgtgcggcggcagttctggtggtgagggtgcgatcgttgggattc gtggtggcgtcatcggtgtaggaacggatatcggtggctcgattcgagtg ccggccgcgttcaacttcctgtacggtctaaggccgagtcatgggcggct gccgtatgcaaagatggcgaacagcatggagggtcaggagacggtgcaca gcgttgtcgggccgattacgcactctgttgagggtgagtccttcgcctct tccttcttttcctgctctataccaggcctccactgtcctcctttcttgct ttttatactatatacgagaccggcagtcactgatgaagtatgttagacct ccgcctcttcaccaaatccgtcctcggtcaggagccatggaaatacgact ccaaggtcatccccatgccctggcgccagtccgagtcggacattattgcc tccaagatcaagaacggcgggctcaatatcggctactacaacttcgacgg caatgtccttccacaccctcctatcctgcgcggcgtggaaaccaccgtcg ccgcactcgccaaagccggtcacaccgtgaccccgtggacgccatacaag cacgatttcggccacgatctcatctcccatatctacgcggctgacggcag cgccgacgtaatgcgcgatatcagtgcatccggcgagccggcgattccaa atatcaaagacctactgaacccgaacatcaaagctgttaacatgaacgag ctctgggacacgcatctccagaagtggaattaccagatggagtaccttga gaaatggcgggaggctgaagaaaaggccgggaaggaactggacgccatca tcgcgccgattacgcctaccgctgcggtacggcatgaccagttccggtac tatgggtatgcctctgtgatcaacctgctggatttcacgagcgtggttgt tccggttacctttgcggataagaacatcgataagaagaatgagagtttca aggcggttagtgagcttgatgccctcgtgcaggaagagtatgatccggag gcgtaccatggggcaccggttgcagtgcaggttatcggacggagactcag tgaagagaggacgttggcgattgcagaggaagtggggaagttgctgggaa atgtggtgactccatagctaataagtgtcagatagcaatttgcacaagaa atcaataccagcaactgtaaataagcgctgaagtgaccatgccatgctac gaaagagcagaaaaaaacctgccgtagaaccgaagagatatgacacgctt ccatctctcaaaggaagaatcccttcagggttgcgtttccagtagtgatt ttaccgctgatgaaatgactggactccctcctcctgctcttatacgaaaa attgcctgactctgcaaaggttgtttgtcttggaagatgatgtgcccccc catcgctcttatctcataccccgccatctttctagattctcatcttcaac aagaggggcaatccatgatctgcgatccagatgtgcttctggcctcatac tctgccttcaggttgatgttcacttaattggtgacgaattcagctgattt gctgcagtatgctttgtgttggttctttccaggcttgtgccagccatgag cgctttgagagcatgttgtcacttataaactcgagtaacggccacatatt gttcactacttgaatcacatacctaattttgatagaattgacatgtttaa agagctgaggtagctttaatgcctctgaagtattgtgacacagcttctca cagagtgagaatgaaaagttggactccccctaatgaagtaaaagtttcgt ctctgaacggtgaagagcatagatccggcatcaactacctggctagacta cgacgtcaattctgcggccttttgacctttatatatgtccattaatgcaa tagattctttttttttttttttttttttttttttttttttttttttttgc ccaatttcgcagatcaaagtggacgttatagcatcataactaagctcagt tgctgagggaagccgtctactaccttagcccatccatccagctccatacc ttgatactttagacgtgaagcaattcacactgtacgtctcgcagctctcc ttcccgctcttgatccccactggggtccatggtgcgtgtatcgtcccctc cacaattctatgccatggtacctccagcttatcaatgccccgctaacaag tcgcctctttgccttgatagcttatcgataaaactttttttccgccagaa aggctccgcccacagacaagaaaaaaaattcaccgcctagcctttggccc cggcatttggctaaacctcgagcctctctcccgtcttggggtatcaggaa gaaaagaaaaaaatccatcgccaagggctgttttggcatcaccacccgaa aacagcacttcctcgatcaaaagttgcccgccatgaagaccacgtggaag gacatccctccggtgcctacgcaccaggagtttctggacattgtgctgag caggacccagcgcaaactgcccactcagatccgtgccggcttcaagatta gcagaattcgaggtacgtcgcattgcccatcgcaggatgtctcattatcg gggtccttggagaacgatcatgattgcatggcgatgctaacacatagaca gccttctacactcgaaaggtcaagttcacccaggagacgttttccgaaaa gttcgcctccatcctcgacagcttccctcgcctccaggacatccacccct tccacaaggaccttctcaacaccctctacgatgccgaccacttcaagatt gcccttggccagatgtccactgccaagcacctggtcgagaccatctcgcg cgactacgtccgtctcttgaaatacgcccagtcgctctaccagtgcaagc agctcaagcgggccgctctcggtcgcatggccacgctggtcaagcgcctc aaggaccccctgctgtacctggaccaggtccgccagcatctcggccgtct tccctccatcgaccccaacaccaggaccctgctcatctgcggttacccca atgttggcaagtccagcttcctgcgaagtatcacccgcgccgatgtggac gtccagccctatgctttcaccaccaagagtctgtttgtcggccactttga ctacaagtacctgcgattccaggccattgatacccccggtattctggacc accctcttgaggagatgaacactatcgaaatgcagaggtatgtggcgcgg cta.

Creation of the Archy3 strain from the Archy2 T. reesei strain. The Archy 2 strain was transformed with a vector to integrate at the same pyr2 locus and replace the hygromycin resistance gene with the coding region of the pyr2 gene. The hygromycin deletion cassette is shown in FIG. 7. This re-introduction of the pyr2 gene back into the pyr2 locus placed it between the T. reesei cbh1 promoter and the partial amdS selectable marker. This strain could be selected for uridine prototrophy and sensitivity to hygromycin. The nucleotide sequence of the hygR knockout cassette is 9088 base pairs in length: bases 1-1994 correspond to the pyr2 3′ homologous region; bases 1995-3497 correspond to the T. reesei cbh1 promoter; bases 3564-5137 correspond to the pyr2 selectable marker; bases 5280-7270 correspond to the A. nidulans amdS 3′ partial marker; bases 7271-9088 correspond to the pyr2 5′ homologous region. The nucleotide sequence of the hygR knockout cassette is provided as SEQ ID NO:16:

atcacgccctcgcataaaagaccctcaagagtccatgtgccctatctgcc tgatcttcctaacccttatttaacattggccctatcacaacctagttctt cttcagcctgctttgtcaacacttgtcacggttcaactcaacgtaatcag caggtagcaggacgaggatagggagagaaacgaagagaagaagaggagag aggaagaaaaaaaaaagaaaagaaagaaaaagggaaaaggaaagaaggag gaaaagagaagaaagtcagatgaagaagcaagaagacgccatggtagcca ccgcttgtcagggctccttagcaacgaacaactctagcttggggacttgt cgatgtgtcgtttccttcctacccatcagcaccaacgatgagttcgatat agacgaggacctcatggaagtagagaccattgggttcgacaggatctctc agtttcacttctatgaggtctgtcgctcggatgactttttgaggagcttc cccttctgcttcaaccccaaactctctttcctgaaaccgcagcacgttgg cacggccgtgttgctggagcagtttgctttcgagcactctcagcgtggtt tcagcagcccactggtgagtggcctcattgacgtccacaccttgctcctg tcgcatgcgtatctggtgggaacgactgctccaaggaggattgctaacga ggttgtaggccgaatatcgcatcagattctccggtaaccttagctacggc ctcttcaacatctgtgacatgacggagcgcaagtactggtggttggcgac caagatgcgcggctggaacatcgacggctgccccgaagacgtcaggagac tcatgtttgttcacatcatcgccaccctgggatgcagccccgtcgtgacg gatgaagacatggactaccccaagaactgggcggcaattctccacggtag agacagatatccgagtgaacctgtgggccaccggcctcatgggcgcacca tctgcctccactcggtggccgtctgccctcgtctccagggcttgggtctc ggtactgcgactctgaagtcgtatgtgcagcgcatgaacagcctcggcgc cgcggaccgtgttgctctcgtttgccgcaagcccgagacgagattttttg aaagatgcggcttcaggaacagcggccggagtagtatcaagactctggtc ggcgaatactacaacatggtgtgtgcttccacatcgacttggccagactc tatacgattttcaaacctcgctatacgtcatattgacttgtttctttagg tcttcgatttgcccgggcccaaagactttatcgactggaatagcattgcc gacgctgccaagaagatgtgaaccatttgactgatacgatgtgtgctacg catgtcgaccttctttgtttgtttctttggcggctctttgtataccttgg gacacggcagacgcatgtctatgtgaagaaaacgttcacggcgctgtttg catcaggaatatgatcattaaacatggagcgtaatggtattaatgatcaa ctagaaaaatggtatggaagggcgagagggcgatcaacaaagcagcccgg ggcatagtctggaagcagcaggaattggaagggaaaaggaagctgcacaa tgaagggatatcgtgagcggagtggctcacgagagtatcaacagactggc gaaagcaagcaattgccaacgccggctattaggccataagatggcctgtt gtgagtcccagttgcacgtatccccatatgactgctctgtcgctgacttg aaaaaaaatagggaggataaaggagaaagaaagtgagacaacccgtgagg gacttggggtagtaggagaacacatgggcaaccgggcaatacacgcgatg tgagacgagttcaacggcgaatggaaaatcttgaaaaacaaaataaaata actgccctccatacgggtatcaaattcaagcagttgtacggaggctagat agagttgtgaagtcggtaatcccgctgtatagtaatacgagtcgcatcta aatactccgaagctgctgcgaacccggagaatcgagatgtgctggaaagc ttctagcgagcggctaaattagcatgaaaggctatgagaaattctggaga cggcttgttgaatcatggcgttccattcttcgacaagcaaagcgttccgt cgcagtagcaggcactcattcccgaaaaaactcggagattcctaagtagc gatggaaccggaataatataataggcaatacattgagttgcctcgacggt tgcaatgcaggggtactgagcttggacataactgttccgtaccccacctc ttctcaacctttggcgtttccctgattcagcgtacccgtacaagtcgtaa tcactattaacccagactgaccggacgtgttttgcccttcatttggagaa ataatgtcattgcgatgtgtaatttgcctgcttgaccgactggggctgtt cgaagcccgaatgtaggattgttatccgaactctgctcgtagaggcatgt tgtgaatctgtgtcgggcaggacacgcctcgaaggttcacggcaagggaa accaccgatagcagtgtctagtagcaacctgtaaagccgcaatgcagcat cactggaaaatacaaaccaatggctaaaagtacataagttaatgcctaaa gaagtcatataccagcggctaataattgtacaatcaagtggctaaacgta ccgtaatttgccaacggcttgtggggttgcagaagcaacggcaaagcccc acttccccacgtttgtttcttcactcagtccaatctcagctggtgatccc ccaattgggtcgcttgtttgttccggtgaagtgaaagaagacagaggtaa gaatgtctgactcggagcgttttgcatacaaccaagggcagtgatggaag acagtgaaatgttgacattcaaggagtatttagccagggatgcttgagtg tatcgtgtaaggaggtttgtctgccgatacgacgaatactgtatagtcac ttctgatgaagtggtccatattgaaatgtaaagtcggcactgaacaggca aaagattgagttgaaactgcctaagatctcgggccctcgggccttcggcc tttgggtgtacatgtttgtgctccgggcaaatgcaaagtgtggtaggatc gaacacactgctgcctttaccaagcagctgagggtatgtgataggcaaat gttcaggggccactgcatggtttcgaatagaaagagaagcttagccaaga acaatagccgataaagatagcctcattaaacggaatgagctagtaggcaa agtcagcgaatgtgtatatataaaggttcgaggtccgtgcctccctcatg ctctccccatctactcatcaactcagatcctccaggagacttgtacacca tcttttgaggcacagaaacccaatagtcaaccgcggactgcgcatcatgt atcggaagttggccgtcatctcggccttcttggccacacctcgtgctaga ctaggcgcgtcaatatgtggccgttactcgagtttataagtgacaacatg ctctcaaagcgctcatggctggcacaagcctggaaagaaccaacacaaag catactgcagcaaatcagctgaattcgtcaccaattaagtgaacatcaac ctgaaggcagagtatgaggccagaagcacatctggatcgcagatcatgga ttgcccctcttgttgaagatgagaatctagaaagatggcggggtatgaga taagagcgatgggggggcacatcatcttccaagacaaacaacctttgcag agtcaggcaatttttcgtataagagcaggaggagggagtccagtcatttc atcagcggtaaaatcactctagacaatcttcaagatgagttctgccttgg gtgacttatagccatcatcatacctagacagaagcttgtgggatactaag accaacgtacaagctcgcactgtacgctttgacttccatgtgaaaactcg atacggcgcgcctctaaattttatagctcaaccactccaatccaacctct gcatccctctcactcgtcctgatctactgttcaaatcagagaataaggac actatccaaatccaacagaatggctaccacctcccagctgcctgcctaca agcaggacttcctcaaatccgccatcgacggcggcgtcctcaagtttggc agcttcgagctcaagtccaagcggatatccccctacttcttcaacgcggg cgaattccacacggcgcgcctcgccggcgccatcgcctccgcctttgcaa agaccatcatcgaggcccaggagaaggccggcctagagttcgacatcgtc ttcggcccggcctacaagggcatcccgctgtgctccgccatcaccatcaa gctcggcgagctggcgccccagaacctggaccgcgtctcctactcgtttg accgcaaggaggccaaggaccacggcgagggcggcaacatcgtcggcgct tcgctcaagggcaagagggtcctgattgtcgacgacgtcatcaccgccgg caccgccaagagggacgccattgagaagatcaccaaggagggcggcatcg tcgccggcatcgtcgtggccctggaccgcatggagaagctccccgctgcg gatggcgacgactccaagcctggaccgagtgccattggcgagctgaggaa ggagtacggcatccccatctttgccatcctcactctggatgacattatcg atggcatgaagggctttgctacccctgaggatatcaagaacacggaggat taccgtgccaagtacaaggcgactgactgattgaggcgttcaatgtcaga agggagagaaagactgaaaaggtggaaagaagaggcaaattgttgttatt attattattctatctcgaatcttctagatcttgtcgtaaataaacaagcg taactagctagcctccgtacaactgcttgaatttgatacccgtatggagg gcagttattttattttgatttcaagattttccattcgccgttgaactcgt ctcacatcgcgtgtattgcccggttgcccatgtgtacgcgtttcgggttt acctcttccagatacagctcatctgcaatgcattaatgcattggacctcg caaccctagtacgcccttcaggctccggcgaagcagaagaatagcttagc agagtctattttcattttcgggagactagcattctgtaaacgggcagcaa tcgcccagcagttagtagggtcccctctacctctcagggagatgtaacaa cgccaccttatgggactatcaagctgacgctggcttctgtgcagacaaac tgcgcccacgagttcttccctgacgccgctctcgcgcaggcaagggaact cgatgaatactacgcaaagcacaagagacccgttggtccactccatggcc tccccatctctctcaaagaccagcttcgagtcaaggtacaccgttgcccc taagtcgttagatgtccctttttgtcagctaacatatgccaccagggcta cgaaacatcaatgggctacatctcatggctaaacaagtacgacgaagggg actcggttctgacaaccatgctccgcaaagccggtgccgtcttctacgtc aagacctctgtcccgcagaccctgatggtctgcgagacagtcaacaacat catcgggcgcaccgtcaacccacgcaacaagaactggtcgtgcggcggca gttctggtggtgagggtgcgatcgttgggattcgtggtggcgtcatcggt gtaggaacggatatcggtggctcgattcgagtgccggccgcgttcaactt cctgtacggtctaaggccgagtcatgggcggctgccgtatgcaaagatgg cgaacagcatggagggtcaggagacggtgcacagcgttgtcgggccgatt acgcactctgttgagggtgagtccttcgcctcttccttcttttcctgctc tataccaggcctccactgtcctcctttcttgctttttatactatatacga gaccggcagtcactgatgaagtatgttagacctccgcctcttcaccaaat ccgtcctcggtcaggagccatggaaatacgactccaaggtcatccccatg ccctggcgccagtccgagtcggacattattgcctccaagatcaagaacgg cgggctcaatatcggctactacaacttcgacggcaatgtccttccacacc ctcctatcctgcgcggcgtggaaaccaccgtcgccgcactcgccaaagcc ggtcacaccgtgaccccgtggacgccatacaagcacgatttcggccacga tctcatctcccatatctacgcggctgacggcagcgccgacgtaatgcgcg atatcagtgcatccggcgagccggcgattccaaatatcaaagacctactg aacccgaacatcaaagctgttaacatgaacgagctctgggacacgcatct ccagaagtggaattaccagatggagtaccttgagaaatggcgggaggctg aagaaaaggccgggaaggaactggacgccatcatcgcgccgattacgcct accgctgcggtacggcatgaccagttccggtactatgggtatgcctctgt gatcaacctgctggatttcacgagcgtggttgttccggttacctttgcgg ataagaacatcgataagaagaatgagagtttcaaggcggttagtgagctt gatgccctcgtgcaggaagagtatgatccggaggcgtaccatggggcacc ggttgcagtgcaggttatcggacggagactcagtgaagagaggacgttgg cgattgcagaggaagtggggaagttgctgggaaatgtggtgactccatag ctaataagtgtcagatagcaatttgcacaagaaatcaataccagcaactg taaataagcgctgaagtgaccatgccatgctacgaaagagcagaaaaaaa cctgccgtagaaccgaagagatatgacacgcttccatctctcaaaggaag aatcccttcagggttgcgtttccagtagtgattttaccgctgatgaaatg actggactccctcctcctgctcttatacgaaaaattgcctgactctgcaa aggttgtttgtcttggaagatgatgtgcccccccatcgctcttatctcat accccgccatctttctagattctcatcttcaacaagaggggcaatccatg atctgcgatccagatgtgcttctggcctcatactctgccttcaggttgat gttcacttaattggtgacgaattcagctgatttgctgcagtatgctttgt gttggttctttccaggcttgtgccagccatgagcgctttgagagcatgtt gtcacttataaactcgagtaacggccacatattgttcactacttgaatca catacctaattttgatagaattgacatgtttaaagagctgaggtagcttt aatgcctctgaagtattgtgacacagcttctcacagagtgagaatgaaaa gttggactccccctaatgaagtaaaagtttcgtctctgaacggtgaagag catagatccggcatcaactacctggctagactacgacgtcaattctgcgg ccttttgacctttatatatgtccattaatgcaatagattctttttttttt tttttttttttttttttttttttttttttttgcccaatttcgcagatcaa agtggacgttatagcatcataactaagctcagttgctgagggaagccgtc tactaccttagcccatccatccagctccataccttgatactttagacgtg aagcaattcacactgtacgtctcgcagctctccttcccgctcttgcttcc ccactggggtccatggtgcgtgtatcgtcccctccacaattctatgccat ggtacctccagcttatcaatgccccgctaacaagtcgcctctttgccttg atagcttatcgataaaactttttttccgccagaaaggctccgcccacaga caagaaaaaaaattcaccgcctagcctttggccccggcatttggctaaac ctcgagcctctctcccgtcttggggtatcaggaagaaaagaaaaaaatcc atcgccaagggctgttttggcatcaccacccgaaaacagcacttcctcga tcaaaagttgcccgccatgaagaccacgtggaaggacatccctccggtgc ctacgcaccaggagtttctggacattgtgctgagcaggacccagcgcaaa ctgcccactcagatccgtgccggcttcaagattagcagaattcgaggtac gtcgcattgcccatcgcaggatgtctcattatcggggtccttggagaacg atcatgattgcatggcgatgctaacacatagacagccttctacactcgaa aggtcaagttcacccaggagacgttttccgaaaagttcgcctccatcctc gacagcttccctcgcctccaggacatccaccccttccacaaggaccttct caacaccctctacgatgccgaccacttcaagattgcccttggccagatgt ccactgccaagcacctggtcgagaccatctcgcgcgactacgtccgtctc ttgaaatacgcccagtcgctctaccagtgcaagcagctcaagcgggccgc tctcggtcgcatggccacgctggtcaagcgcctcaaggaccccctgctgt acctggaccaggtccgccagcatctcggccgtcttccctccatcgacccc aacaccaggaccctgctcatctgcggttaccccaatgttggcaagtccag cttcctgcgaagtatcacccgcgccgatgtggacgtccagccctatgctt tcaccaccaagagtctgtttgtcggccactttgactacaagtacctgcga ttccaggccattgatacccccggtattctggaccaccctcttgaggagat gaacactatcgaaatgcagaggtatgtggcgcggct.

Creation of the A5D strain from the Archy3 T. reesei strain. Native T. reesei bgl1 was deleted from the Archy 3 strain using a double recombination vector known in the art. Hygromycin resistance was used as the selectable marker for bgl1 deletion. In addition, the hygromycin resistance marker was flanked by loxP sites. The deletion cassette is shown in FIG. 8. Subsequent hygromycin resistant transformants were analyzed for bgl1 deletion. A strain confirmed for deletion of bgl1 was next transformed with a telomeric vector encoding cre recombinase and a functional amdS for selection of transformants to facilitate removal of the hygromycin by cre recombinase expression and loop out of the loxP sites. The telomeric vector encoding the cre recombinase is schematically shown in FIG. 10. Transformants were first obtained on acetamide media, then transferred to potato dextrose agar and replica plated onto hygromycin media to screen for hygromycin sensitivity. Strains sensitive to hygromycin, were again transferred to potato dextrose media and replica plated to acetamide media. A strain was selected which tossed the ability to grow on acetamide, indicative of a loss of the telomeric vector. The nucleotide sequence of the bgl1 knockout cassette is provided as SEQ ID NO:17:

aatggtaggaatgctgggatataggctctgtgctggcaagttgatggatc ctcgaatgaggccgccctgcaaggggaacatcagagatctaccattgcct ccttggcccaatccactatcatacctacctcatgatcattcctgcgaagg tctaccagtaaatatttcctcgtcccgtgtttcatcatgtccagaacctc atctcgccaaattgactttgccacagtgtctggagctgggtaagcagcgt gccaaggaattgttgtcgagtctgtgccaggcattgtgcccgacattgtg aacttcagccaggagaacttttcgatcgcacctatgctgagcaccgtggg catgcgatggcttcaataatgcagttcgagagggagtgtgtcatgcccta aagctcattggccacctccacaggctagctctacctgcatctgtagatgg actttccttgtcctcctccttcagaaaacctcttggtcgctcgcaggtaa ctgttgttgccgtcattgtttgacagtggatagccaaggcaaaaccgtct gctttcaacggaagcattcggcggttgtttgtcatcgtgttatcgatcga ccaggagaacccagacgagtgttgttcgagagaatcatcgacgatgtgaa gaggcgacgactagtatctagaagattataatcgaacaaatcagcgtttg tctgtcgggcgtttgagggcgcagttgcccgccaaagcagcgtcgcaata tataggcagcgagagactgtcaacagccagccgccatgtgatcgatcgta gccgtcttcccgatcttccctaaacccctttctttggggggcgggggcag cggcgttctaatatttgctggctgtctggataacgtgaatggtagacatg gtaatgttcggtctgcgaaacatttgtacaattggagtttacgatcgaga tggaaggaaacgctccacaaactcggtgactgggttgccatcaggtgctc agggcatagcgttctctgcaaatagaggaaagagaatagcactagtgaaa gtgtgaatcacaatgaagaggaggttgttgccggaatgctttgagcagcg tcaaagttgaacttgaagctatcacaaattgcagggtaaagtacatgttg gtgccagtttgacagcacagtgcgcggagcggaggatgtcgcggaagagg cgcgacgctaacccgggccttcttctcagtgagcagaactcctgctgcaa gagttccttctctctgcgagatgacgtgaggcccaatttgcagcttccct cgaacaaggtgattgaacatctctcttccctcacatttcatcatcactac ctcctcaattcacttctgcttcggccgtcttcatcattcatgttactgct ctgatgcctatcctgaagattgtattcctgcagtattcacgccatcccac cttcggtcctcactcacagtcacaggtcaaccgccttcaccctcctcgcg atgatgtcggcaatctggtggatcaatgtgcggttgagggccgccgtagt gaggatgggcatggggaacgaggtcgcccattcgcccacagataacttcg tatagcatacattatacgaagttatcctgggcttgtgactggtcgcgagc tgccactaagtggggcagtaccattttatcggacccatccagctatggga cccactcgcaaatttttacatcattttctttttgctcagtaacggccacc ttttgtaaagcgtaaccagcaaacaaattgcaattggcccgtagcaaggt agtcagggcttatcgtgatggaggagaaggctatatcagcctcaaaaata tgttgccagctggcggaagcccggaaggtaagtggattcttcgccgtggc tggagcaaccggtggattccagcgtctccgacttggactgagcaattcag cgtcacggattcacgatagacagctcagaccgctccacggctggcggcat tattggttaacccggaaactcagtctccttggccccgtcccgaagggacc cgacttaccaggctgggaaagccagggatagaatacactgtacgggcttc gtacgggaggttcggcgtagggttgttcccaagttttacacaccccccaa gacagctagcgcacgaaagacgcggagggtttggtgaaaaaagggcgaaa attaagcgggagacgtatttaggtgctagggccggtttcctccccatttt tcttcggttccctttctctcctggaagactttctctctctctcttcttct cttcttccatcctcagtccatcttcctttcccatcatccatctcctcacc tccatctcaactccatcacatcacaatcgatatgaaaaagcctgaactca ccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctcc gacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcga tgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtt tctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccg attccggaagtgcttgacattggggaattcagcgagagcctgacctattg catctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccg aactgcccgctgttctgcagccggtcgcggaggccatggatgcgatcgct gcggccgatcttagccagacgagcgggttcggcccattcggaccgcaagg aatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatc cccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtcc gtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccga agtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacgg acaatggccgcataacagcggtcattgactggagcgaggcgatgttcggg gattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggc ttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttg caggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaa ctctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgca gggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgta cacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaa gtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaa ggaatagagtagatgccgaccgggatccacttaacgttactgaaatcatc aaacagcttgacgaatctggatataagatcgttggtgtcgatgtcagctc cggagttgagacaaatggtgttcaggatctcgataagatacgttcatttg tccaagcagcaaagagtgccttctagtgatttaatagctccatgtcaaca agaataaaacgcgtttcgggtttacctcttccagatacagctcatctgca atgcattaatgcattggacctcgcaaccctagtacgcccttcaggctccg gcgaagcagaagaatagcttagcagagtctattttcattttcgggagacg agatcaagcagatcaacggtcgtcaagagacctacgagactgaggaatcc gctcttggctccacgcgactatatatttgtctctaattgtactttgacat gctcctcttctttactctgatagcttgactatgaaaattccgtcaccagc ccctgggttcgcaaagataattgcactgtttcttccttgaactctcaagc ctacaggacacacattcatcgtaggtataaacctcgaaaatcattcctac taagatgggtatacaatagtaaccatggttgcctagtgaatgctccgtaa cacccaatacgccggccgataacttcgtatagcatacattatacgaagtt atacttggcgcgcctagtggaacacgagcacataagcttttacctatggt tatcgcttgcatctacgcgccgttgatggtggaggatggtggacgttccc gagacccctacgagctgtggcatcgtcaaactgtgcccacagacctttgt cttgctttcataacctcgaggagtgtttccagactcatcatccatacaca agcagtattaatcaaagaaactcggtcgcaatggcaaaaatggtttgcaa acagaaaactatggcctcttcctattccatcattaactactctacccgtt tgtcataacaacatcattaaaacccttatgcgtcaggtgtagcatccttg atctgttgctcctccaacggccagttctcaatcgttacctcttctcccac caactcaaactcaagcttcacagactcgtcggtgttcaaggctagctcat acttgccggggtatacaatccggtttccgtgagaatcaacacgggcgaga gcactgacagggatggggatgctgagcttggaagagtgaccaggcttgat gtcggcaagtcggtcgaatccgacgagccacttgttcgggtacggggctg ggccagcgttgcttgtgcgaacaaacagcatggccgtatatggggactcc gtcttgcccgagttcttgatgttggcctcgaaggtgaagacgggaatctg ctcgctgtaagtgtatccggggtgaggagcagagaggatcgatgaggtgt taacttgaggctcttggggtggctggcgagagtctccttgaaggtggtgt agaagagaccactgccaaactcgtagacgggtttgccggtgtaccagatg taagtctgtccagggtttgactttccatcgggtcggaggttcatgtcatt ctgggggaattggtgaacatactcagccgggtactgagtggtgaccagtc ggccggcaggagcacgcttgccagagagaatgtcgaagagggcaacgcct cccgactggccgggatatccgccccagacgagggagttgaccttcttgtt gctcttgagcgaggatgagtctacctgaccaccgcccatttgcaggacga caaggggtttgccgacctcgctgagctgcttgatgagatccagctgatta ccgggccaagcaatgtccgtgcggtcagcgccctcctgttcaatggtgtt gtcaattccaccgaggtagatgatggcatccgacttcttggcggcagcaa tggccttggcaaagccagtggtgctgttgccggcgatctctgtgccgagt tcaaagttgacgtgatagccggccttcttagcagcttccagagggctgat gaggtatggggcagggccatagtagttgccttgcatttgggttgtggcat tggcccatggtccgatcagagcaatgctgcgcaccttcttggacagaggg agagtgccatcgttcttgagcaggacgatgccctcaacagcagcctcgta cgagatgtt.

The nucleotide sequence of the telomeric vector, pTTT-cre, is provided as SEQ ID NO:18:

ttgtacaaagtggtgatcgcgccgcgcgccagctccgtgcgaaagcctga cgcaccggtagattcttggtgagcccgtatcatgacggcggcgggagcta catggccccgggtgatttattttttttgtatctacttctgacccttttca aatatacggtcaactcatctttcactggagatgcggcctgcttggtattg cgatgttgtcagcttggcaaattgtggctttcgaaaacacaaaacgattc cttagtagccatgcattttaagataacggaatagaagaaagaggaaatta aaaaaaaaaaaaaaacaaacatcccgttcataacccgtagaatcgccgct cttcgtgtatcccagtaccagtttattttgaatagctcgcccgctggaga gcatcctgaatgcaagtaacaaccgtagaggctgacacggcaggtgttgc tagggagcgtcgtgttctacaaggccagacgtcttcgcggttgatatata tgtatgtttgactgcaggctgctcagcgacgacagtcaagttcgccctcg ctgcttgtgcaataatcgcagtggggaagccacaccgtgactcccatctt tcagtaaagctctgttggtgtttatcagcaatacacgtaatttaaactcg ttagcatggggctgatagcttaattaccgtttaccagtgccatggttctg cagctttccttggcccgtaaaattcggcgaagccagccaatcaccagcta ggcaccagctaaaccctataattagtctcttatcaacaccatccgctccc ccgggatcaatgaggagaatgagggggatgcggggctaaagaagcctaca taaccctcatgccaactcccagtttacactcgtcgagccaacatcctgac tataagctaacacagaatgcctcaatcctgggaagaactggccgtgataa gcgcgcccgcctcgcaaaaaccatccctgatgaatggaaagtccagacgc tgcctgcggaagacagcgttattgatttcccaaagaaatcggggatcctt tcagaggccgaactgaagatcacagaggcctccgctgcagatcttgtgtc caagctggcggccggagagttgacctcggtggaagttacgctagcattct gtaaacgggcagcaatcgcccagcagttagtagggtcccctctacctctc agggagatgtaacaacgccaccttatgggactatcaagctgacgctggct tctgtgcagacaaactgcgcccacgagttcttccctgacgccgctctcgc gcaggcaagggaactcgatgaatactacgcaaagcacaagagacccgttg gtccactccatggcctccccatctctctcaaagaccagcttcgagtcaag gtacaccgttgcccctaagtcgttagatgtccctttttgtcagctaacat atgccaccagggctacgaaacatcaatgggctacatctcatggctaaaca agtacgacgaaggggactcggttctgacaaccatgctccgcaaagccggt gccgtcttctacgtcaagacctctgtcccgcagaccctgatggtctgcga gacagtcaacaacatcatcgggcgcaccgtcaacccacgcaacaagaact ggtcgtgcggcggcagttctggtggtgagggtgcgatcgttgggattcgt ggtggcgtcatcggtgtaggaacggatatcggtggctcgattcgagtgcc ggccgcgttcaacttcctgtacggtctaaggccgagtcatgggcggctgc cgtatgcaaagatggcgaacagcatggagggtcaggagacggtgcacagc gttgtcgggccgattacgcactctgttgagggtgagtccttcgcctcttc cttcttttcctgctctataccaggcctccactgtcctcctttcttgcttt ttatactatatacgagaccggcagtcactgatgaagtatgttagacctcc gcctcttcaccaaatccgtcctcggtcaggagccatggaaatacgactcc aaggtcatccccatgccctggcgccagtccgagtcggacattattgcctc caagatcaagaacggcgggctcaatatcggctactacaacttcgacggca atgtccttccacaccctcctatcctgcgcggcgtggaaaccaccgtcgcc gcactcgccaaagccggtcacaccgtgaccccgtggacgccatacaagca cgatttcggccacgatctcatctcccatatctacgcggctgacggcagcg ccgacgtaatgcgcgatatcagtgcatccggcgagccggcgattccaaat atcaaagacctactgaacccgaacatcaaagctgttaacatgaacgagct ctgggacacgcatctccagaagtggaattaccagatggagtaccttgaga aatggcgggaggctgaagaaaaggccgggaaggaactggacgccatcatc gcgccgattacgcctaccgctgcggtacggcatgaccagttccggtacta tgggtatgcctctgtgatcaacctgctggatttcacgagcgtggttgttc cggttacctttgcggataagaacatcgataagaagaatgagagtttcaag gcggttagtgagcttgatgccctcgtgcaggaagagtatgatccggaggc gtaccatggggcaccggttgcagtgcaggttatcggacggagactcagtg aagagaggacgttggcgattgcagaggaagtggggaagttgctgggaaat gtggtgactccatagctaataagtgtcagatagcaatttgcacaagaaat caataccagcaactgtaaataagcgctgaagtgaccatgccatgctacga aagagcagaaaaaaacctgccgtagaaccgaagagatatgacacgcttcc atctctcaaaggaagaatcccttcagggttgcgtttccagtctagacacg tataacggcacaagtgtctctcaccaaatgggttatatctcaaatgtgat ctaaggatggaaagcccagaatatcgatcgcgcgcagatccatatatagg gcccgggttataattacctcaggaaatagctttaagtagcttattaagta ttaaaattatatatatttttaatataactatatttctttaataaataggt attttaagctttatatataaatataataataaaataatatattatatagc tttttattaataaataaaatagctaaaaatataaaaaaaatagctttaaa atacttattataattagaattttatatatttttaatatataagatctttt acttttttataagcttcctaccttaaattaaatttttacttttttttact attttactatatcttaaataaaggctttaaaaatataaaaaaaatcttct tatatattataagctataaggattatatatatatttttttttaattttta aagtaagtattaaagctagaattaaagttttaattttttaaggctttatt taaaaaaaggcagtaatagcttataaaagaaatttctttttcttttatac taaaagtactttttttttaataaggttagggttagggttagggttagggt tagggttagggttagggttagggttagggttagggttagggttagggtta gggttagggttagggtaagggtttaaacaaagccacgttgtgtctcaaaa tctctgatgttacattgcacaagataaaaatatatcatcatgaacaataa aactgtctgcttacataaacagtaatacaaggggtgttatgagccatatt caacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgc tgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtg cgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctg aaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcag actaaactggctgacggaatttatgcctcttccgaccatcaagcatttta tccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaa acagcattccaggtattagaagaatatcctgattcaggtgaaaatattgt tgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgta attgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatca cgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaa tggctggcctgagaacaagtctggaaagaaatgcataagcttttgccatt ctcaccggattcagtcgtcactcatggtgatttctcacttgataacctta tttttgacgaggggaaattaataggttgtattgatgttggacgagtcgga atcgcagaccgataccaggatcttgccatcctatggaactgcctcggtga gttttctccttcattacagaaacggctttttcaaaaatatggtattgata atcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttc taatcagaattggttaattggttgtaacactggcagagcattacgctgac ttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaa ggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagc aaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaac cgtggctccctcactttctggctggatgatggggcgattcaggcctggta tgagtcagcaacaccttcttcacgaggcagacctcagcggtttaaaccta accctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaac cctaaccctaaccctaaccctaaccctaaccctaacctaaccctaatggg gtcgatctgaaccgaggatgagggttctatagactaatctacaggccgta catggtgtgattgcagatgcgacgggcaaggtgtacagtgtccagaagga ggagagcggcataggtattgtaatagaccagctttacataataatcgcct gttgctactgactgatgaccttcttccctaaccagtttcctaattaccac tgcagtgaggataaccctaactcgctctggggttattattatactgatta gcaggtggcttatatagtgctgaagtactataagagtttctgcgggagga ggtggaaggactataaactggacacagttagggatagagtgatgacaaga cctgaatgttatcctccggtgtggtatagcgaattggctgaccttgcaga tggtaatggtttaggcagggtttttgcagagggggacgagaacgcgttct gcgatttaacggctgctgccgccaagctttacggttctctaatgggcggc cgcctcaggtcgacgtcccatggccattcgaattcgtaatcatggtcata gctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatac gagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaa ctcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacct gtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtt tgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcg gtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacg gttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaag gccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttc cataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtca gaggtggcgaaacccgacaggactataaagataccaggcgtttccccctg gaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatac ctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacg ctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtg tgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagc cactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagt tcttgaagtggtggcctaactacggctacactagaagaacagtatttggt atctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctc ttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatc ttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaatt aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtct gacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct atttcgttcatccatagttgcctgactccccgtcgtgtagataactacga tacgggagggcttaccatctggccccagtgctgcaatgataccgcgagac ccacgctcaccggctccagatttatcagcaataaaccagccagccggaag ggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtcta ttaattgttgccgggaagctagagtaagtagttcgccagttaatagtttg cgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtt tggtatggcttcattcagctccggttcccaacgatcaaggcgagttacat gatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatc gttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagc actgcataattctcttactgtcatgccatccgtaagatgcttttctgtga ctggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccg agttgctcttgcccggcgtcaatacgggataataccgcgccacatagcag aactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactct caaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgca cccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagc aaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacgga aatgttgaatactcatactcttcctttttcaatattattgaagcatttat cagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaa taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacg tctaagaaaccattattatcatgacattaacctataaaaataggcgtatc acgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctg acacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccg ggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcgg ggctggcttaactatgcggcatcagagcagattgtactgagagtgcacca taaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgtt aaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttat aaatcaaaagaatagcccgagatagggttgagtgttgttccagtttggaa caagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaa ccgtctatcagggcgatggcccactacgtgaaccatcacccaaatcaagt tttaggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagc ccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaagga agggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcgg tcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacag ggcgcgtactatggttgctttgacgtatgcggtgtgaaataccgcacaga tgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcg caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagc tggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccaggg ttttcccagtcacgacgttgtaaaacgacggccagtgcccaagcttacta gatgcatgctcgagcggccgccagtgtgatggatatctgcagaattcgcc cttgactagtgctctctatcctggtggcaggcgtcaagtacccagaggca gcagcgggcttaggagcggcctgggttgttctccgcaccctctacatgct gggctatatttatagcgacaagccgaacggcaccggcaggtacaatggtt cgctgtacttgcttgcgcaagcgggtctttggggattgagcgcatttggt gttgcaaaggatttgatgtaaatgtagtcgacatcttagcacagagggga gagttgataaaatgtggtctgtttgaatgatagtcgggttcgtgacctat attcgtgatagtggagataggtctgcgcctatcttatcgggccggagcaa aaattccaccgcagcggggtgagttttcgttatacagccatcccacttcc agcttcaaattgtcagtttaatccagcccaattcaatcattggagaaccg ccatcatgtcttcgaagtcccacctcccctacgcaattcgcgcaaccaac catcccaaccctttaacatctaaactcttctccatcgccgaggagaagaa aaccaacgtcaccgtctccgcagacgttactacttccgccgagctcctcg atcttgctgaccgtacatcctgcaccaatgcccctccaggataacaaata gctgatgcgtagtgagtacaggcctaggcccctatatcgcagttctgaaa acccacatcgacatcctcaccgatctcaccccgtcgaccctttcctcgct ccaatccctcgcgacaaagcacaacttcctcatctttgaggaccgcaagt tcatcgacatcggcaacaccgtgcaaaagcagtaccacggtggcgctctc cgcatctccgaatgggcacacatcatcaactgcgccatcctgccgggcga agggatcgtcgaggccctcgcacagacaaccaagtctcctgactttaaag acgcgaatcaacgaggtctcctgattcttgccgagatgacgagtaaggga tctcttgcgacaggggagtcacaggcacgctcggttgagtacgcgcggaa gtataaggggtttgtgatgggattcgtgagtacaagggcgttgagtgagg tgctgcccgaacagaaagaggagagcgaggattttgtcgtctttacgact ggggtgaatctgtcggataagggggataagctggggcagcagtatcagac acctgggtcggcggttgggcgaggtgcggactttatcattgcgggtaggg gcatctataaggcggacgatccagtcgaggcggttcagaggtaccgggag gaaggctggaaagcttacgagaaaagagttggactttgagtgtgagtgga aatgtgtaacggtattgactaaaagggatccatatgtttattgcagccag catagtattaccagaaagagcctcactgacggctctagtagtattcgaac agatattattgtgaccagctctgaacgatatgctccctaatctggtagac aagcactgatctaccccttggaacgcagcatctaggctctggctgtgctc taaccctaactagacgattgatcgcagaccatccaatactgaaaagtctc tatcagaggaaatccccaacattgtagtagtcaggttcctttgtggctgg gagagaattggttcgctccactgattccagttgagaaagtgggctagaaa aaagtcttgaagattggagttgggctgtggttatctagtacttctcgagc tctgtacatgtccggtcgcgacgtacgcgtatcgatggcgccagctgcag gcggccgcctgcagccacttgcagtcccgtggaattctcacggtgaatgt aggccttttgtagggtaggaattgtcactcaagcacccccaacctccatt acgcctcccccatagagttcccaatcagtgagtcatggcactgttctcaa atagattggggagaagttgacttccgcccagagctgaaggtcgcacaacc gcatgatatagggtcggcaacggcaaaaaagcacgtggctcaccgaaaag caagatgtttgcgatctaacatccaggaacctggatacatccatcatcac gcacgaccactttgatctgctggtaaactcgtattcgccctaaaccgaag tgcgtggtaaatctacacgtgggcccctttcggtatactgcgtgtgtctt ctctaggtgccattcttttcccttcctctagtgttgaattgtttgtgttg gagtccgagctgtaactacctctgaatctctggagaatggtggactaacg actaccgtgcacctgcatcatgtatataatagtgatcctgagaagggggg tttggagcaatgtgggactttgatggtcatcaaacaaagaacgaagacgc ctcttttgcaaagttttgtttcggctacggtgaagaactggatacttgtt gtgtcttctgtgtatttttgtggcaacaagaggccagagacaatctattc aaacaccaagcttgctcttttgagctacaagaacctgtggggtatatatc tagagttgtgaagtcggtaatcccgctgtatagtaatacgagtcgcatct aaatactccgaagctgctgcgaacccggagaatcgagatgtgctggaaag cttctagcgagcggctaaattagcatgaaaggctatgagaaattctggag acggcttgttgaatcatggcgttccattatcgacaagcaaagcgttccgt cgcagtagcaggcactcattcccgaaaaaactcggagattcctaagtagc gatggaaccggaataatataataggcaatacattgagttgcctcgacggt tgcaatgcaggggtactgagcttggacataactgttccgtaccccacctc ttctcaacctttggcgtttccctgattcagcgtacccgtacaagtcgtaa tcactattaacccagactgaccggacgtgttttgcccttcatttggagaa ataatgtcattgcgatgtgtaatttgcctgcttgaccgactggggctgtt cgaagcccgaatgtaggattgttatccgaactctgctcgtagaggcatgt tgtgaatctgtgtcgggcaggacacgcctcgaaggttcacggcaagggaa accaccgatagcagtgtctagtagcaacctgtaaagccgcaatgcagcat cactggaaaatacaaaccaatggctaaaagtacataagttaatgcctaaa gaagtcatataccagcggctaataattgtacaatcaagtggctaaacgta ccgtaatttgccaacggcttgtggggttgcagaagcaacggcaaagcccc acttccccacgtttgtttcttcactcagtccaatctcagctggtgatccc ccaattgggtcgcttgtttgttccggtgaagtgaaagaagacagaggtaa gaatgtctgactcggagcgttttgcatacaaccaagggcagtgatggaag acagtgaaatgttgacattcaaggagtatttagccagggatgcttgagtg tatcgtgtaaggaggtttgtctgccgatacgacgaatactgtatagtcac ttctgatgaagtggtccatattgaaatgtaaagtcggcactgaacaggca aaagattgagttgaaactgcctaagatctcgggccctcgggccttcggcc tttgggtgtacatgtttgtgctccgggcaaatgcaaagtgtggtaggatc gaacacactgctgcctttaccaagcagctgagggtatgtgataggcaaat gttcaggggccactgcatggtttcgaatagaaagagaagcttagccaaga acaatagccgataaagatagcctcattaaacggaatgagctagtaggcaa agtcagcgaatgtgtatatataaaggttcgaggtccgtgcctccctcatg ctctccccatctactcatcaactcagatcctccaggagacttgtacacca tcttttgaggcacagaaacccaatagtcaaccatcacaagtttgtacaaa aaagcaggctcaccatgagcaacctgctcaccgtccaccagaacctccct gccctccctgtcgacgccacctctgacgaggtccgcaagaacctcatgga catgttccgcgaccgccaggcctttagcgagcacacctggaagatgctcc tcagcgtctgccgatcttgggccgcctggtgcaagctcaacaaccgcaag tggttccccgccgagccggaggacgtccgcgactacctcctctacctgca ggcccgaggcctggccgtcaagaccatccagcagcacctcggccagctca acatgctccaccgacgctctggcctgcctcgccctagcgactctaacgcc gtcagcctggtcatgcgccgcatccgcaaggagaacgtcgacgctggcga gcgagccaagcaggccctcgccttcgagcgcaccgacttcgaccaggtcc gcagcctcatggagaacagcgaccgctgccaggatatccgcaacctcgcc tttctcggcattgcctacaacaccctgctccgcattgccgagatcgcccg catccgcgtcaaggacatctctcgcaccgacggcggccgcatgctcattc acatcggccgcaccaagaccctcgtgtctaccgccggcgtcgagaaggcc ctcagcctcggcgtcaccaagctcgtcgagcgctggatttctgtctccgg cgtcgctgacgaccccaacaactacctcttctgccgcgtccgaaagaacg gcgtcgccgccccttctgccacctctcagctcagcacccgagccctggag ggcatctttgaggccacccaccgcctcatctacggcgccaaggacgactc tggccagcgctacctcgcctggtctggccactctgcccgagtcggcgctg cccgagacatggcccgagccggcgtcagcatccccgagattatgcaggcc ggcggctggaccaacgtcaacatcgtcatgaactacatccgcaccctcga ctctgagaccggcgccatggtccgactcctcgaggacggcgactaaaccc agctttc.

Creation of the MAD6 strain from A5D T. reesei strain. Native egl3 was deleted from the A5D strain using the method previously described for bgl1 deletion. A schematic of the deletion cassette is shown in FIG. 9. Hygromycin resistance was used as the selectable marker for egl3 deletion. The hygromycin resistance marker was flanked by loxP sites. A transformant was confirmed with a deletion of the egl3. The hygromycin marker was removed from this strain as described for creation of the A5D strain. The nucleotide sequence of the egl3 deletion cassette is provided as SEQ ID NO:19:

gggaggtaggcgcagatacggtgcatgggacccgaacccgtaaccggaac acgaccttatcagccctccaactcacaccctctcgcctatcactatccta gatagttcatcggccaactcatgtaacctagctacctacctacctggtaa gaatgcgggctatcatgtctcacggcgcggtacatgtcggtatctcgctg cttccccgcaggttgacgtcggaatccatgcaagtactccctgaaatcga gacgacagagagaacaaccaacgcgcttaaacgcttcatgttcatctaag aggcacattcgaagaactagcttaacacactagacctggcttttcgaccc cctccgcagaaagccgttttctcctcaatcctcccgggcttggcttttgt cagtccgtacttgctgcgctaacagagtcttggacgcagcgtttgcgcat cagtcttgcaggcggttcacgggactaggacaacaggggatgtgacaggc cggatagtaattatgggttatccggggtaagcagggaatttacgaggccg ctttacgtgggggaacagccacttgcggggggaagaggagtagtaggcga ctcggtcgatgagctcgaggtgtctggttgacttggactgcagagcgtag gtaattgagatcgggcaacattatcggtgttcggctcggtatggccgagt tgcgactgcttggtcattcggcgaagctgatgtcgtggtatcctgaagca tcgatatcggaaaccatgatggtcagtctatctgacgtgtgcggtgacaa gcgagtccggattttgtgacatgacgttcaacttcagtcaatgccttagg gctcgataagattaagattgggactggcagcggtctagaacaccgccaca aattctgtccattgaggagcgtgatgtctaggcgcatcactaacacggag ctgtatgaccggcagctcaacggacttctcttcgttcaacggcagtctat ttgcggtacacgaatggatctttcttcctggtcttgaagtgccgcagtgg cgtgcgaatgtatagatgtctcgctacctagaaaagctggcttttctgac agggtcccttccacctctcctaccaacgacaaactgaacaagtatctggc ggtttcccaacgccgaataggccagtcgccaatactccctccagccctga ttgggcccctcgaagtatcgccatgtctgtgtgttgagattattcgatgg acgtcactcccccaacctacaggaagagcaaaatgggagcagtgttctgc aatgagctatataatagatcgctcgatctcatacaaattgtatgctcagt caatacaacgagcggttccaagatcccttctccaacgaccctcgaaacat tgcaacccggtgcagcctgaacttgttcgtatagcctagaaagcgacgcc atcttcatcttttacgcgattagcctcatggctatttgtgccgaagtggg agttgtatggtagcagtgaggagattgtggctacgacacaggcgggttct cttgagcggcttacatctccgcattaggcctgcgtacgatccagatcatg ggaaactttacaatggcttactcgttttatctcaacactgagcttccaat tcactctatgcattgattaacacgtttggtcatgtggttcttcagctgta aatcttcagcttcccaagaattgcaacctcgctgattgctaatagtgttg catgcgttgcatcctggtgcggcagtgcaaaggagagtcaaagtagccgg cagattaatttaagcttatatcactcaggggtaaacagccgtaaaggacc ttttgatctaacatgccgatgtgtatgtagatcacgcaatgcccaccata tcttggcagtcagatttgtccgtggcgcgccaagtataacttcgtataat gtatgctatacgaagttatcggccggcgtattgggtgttacggagcattc actaggcaaccatggttactattgtatacccatcttagtaggaatgattt tcgaggtttatacctacgatgaatgtgtgtcctgtaggcttgagagttca aggaagaaacagtgcaattatctttgcgaacccaggggctggtgacggaa ttttcatagtcaagctatcagagtaaagaagaggagcatgtcaaagtaca attagagacaaatatatagtcgcgtggagccaagagcggattcctcagtc tcgtaggtctcttgacgaccgttgatctgcttgatctcgtctcccgaaaa tgaaaatagactctgctaagctattcttctgcttcgccggagcctgaagg gcgtactagggttgcgaggtccaatgcattaatgcattgcagatgagctg tatctggaagaggtaaacccgaaacgcgttttattcttgttgacatggag ctattaaatcactagaaggcactctttgctgcttggacaaatgaacgtat cttatcgagatcctgaacaccatttgtctcaactccggagctgacatcga caccaacgatcttatatccagattcgtcaagctgtttgatgatttcagta acgttaagtggatcccggtcggcatctactctattcctttgccctcggac gagtgctggggcgtcggtttccactatcggcgagtacttctacacagcca tcggtccagacggccgcgcttctgcgggcgatttgtgtacgcccgacagt cccggctccggatcggacgattgcgtcgcatcgaccctgcgcccaagctg catcatcgaaattgccgtcaaccaagctctgatagagttggtcaagacca atgcggagcatatacgcccggagccgcggcgatcctgcaagctccggatg cctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcc tccagaagaagatgttggcgacctcgtattgggaatccccgaacatcgcc tcgctccagtcaatgaccgctgttatgcggccattgtccgtcaggacatt gttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcct cggcccaaagcatcagctcatcgagagcctgcgcgacggacgcactgacg gtgtcgtccatcacagtttgccagtgatacacatggggatcagcaatcgc gcatatgaaatcacgccatgtagtgtattgaccgattccttgcggtccga atgggccgaacccgctcgtctggctaagatcggccgcagcgatcgcatcc atggcctccgcgaccggctgcagaacagcgggcagttcggtttcaggcag gtcttgcaacgtgacaccctgtgcacggcgggagatgcaataggtcaggc tctcgctgaattccccaatgtcaagcacttccggaatcgggagcgcggcc gatgcaaagtgccgataaacataacgatctttgtagaaaccatcggcgca gctatttacccgcaggacatatccacgccctcctacatcgaagctgaaag cacgagattcttcgccctccgagagctgcatcaggtcggagacgctgtcg aacttttcgatcagaaacttctcgacagacgtcgcggtgagttcaggctt tttcatatcgattgtgatgtgatggagttgagatggaggtgaggagatgg atgatgggaaaggaagatggactgaggatggaagaagagaagaagagaga gagagaaagtcttccaggagagaaagggaaccgaagaaaaatggggagga aaccggccctagcacctaaatacgtctcccgcttaattttcgcccttttt tcaccaaaccctccgcgtctttcgtgcgctagctgtcttggggggtgtgt aaaacttgggaacaaccctacgccgaacctcccgtacgaagcccgtacag tgtattctatccctggctttcccagcctggtaagtcgggtcccttcggga cggggccaaggagactgagtttccgggttaaccaataatgccgccagccg tggagcggtctgagctgtctatcgtgaatccgtgacgctgaattgctcag tccaagtcggagacgctggaatccaccggttgctccagccacggcgaaga atccacttaccttccgggcttccgccagctggcaacatatttttgaggct gatatagccttctcctccatcacgataagccctgactaccttgctacggg ccaattgcaatttgtttgctggttacgctttacaaaaggtggccgttact gagcaaaaagaaaatgatgtaaaaatttgcgagtgggtcccatagctgga tgggtccgataaaatggtactgccccacttagtggcagctcgcgaccagt cacaagcccaggataacttcgtataatgtatgctatacgaagttatctgt gggcgttatgaataatagactggaaccgggccctttgattgacgactcca tattttgtagatgtagcaactcggcaagagcattatgtgcaatacatttg ttaccatacaaaggcagctgccagacgacttgtattgcgtacaattctca cggcaagctttccaggtgttatgcattatgcgcaaatgcttgatgcttac cgcaggattaatctcggaagaagcgctgcaagctatatgggtgtagtaga tatgtagatgtaccaaccaatgaagaacatttatggtctagaatgtagtg atgaaggttttgagtaatttgtatcaagtaagacgatattattgatataa taccaagcatatattcatgataaattacttggaaccacccttgcgtccgg cctcacgagccttctcactgccgggctcgaaggagccactggaggcctgt ccacccttggatgcgatttcctgcaccttttccttgggcctgcacgtcga ttagacatgattcaaatcgagatcttggaatatcttacatgctggcgaag ccaccggtgtggctggactgtccgcccttctgcgcaatgctttgaacctc ctccttggggctgtgtagaaaggtttgttagcaacattactacaactctc aggactcggtggtcgtaccggttggcgaagtttccggggttatcgttgcc agacattgtgtgattatttggtgtgcaaatgtgtgctatgtgtgttgttg ctgttggtgatgatgctgaagctgttgaaagcaggctggttctgtgggag agacttgggatatttatatccaaagttcggtcgtgttccttctggaagct cttctctactccatacaatcatccaaagttgtcgtcattgagcgttgatc agtagtagcctctgaggtcatcaccatgatccttccggccaacagtcggc actcatcaacagcaacaatcagccgccacaaacataggtacagtaaggag ttagatatcatgtagtcgtcgagtactcgacatcatgacgtacaagcttt gccagtgtcggtaggtgcaagtatgatgatcgtatccgccgttgttcgat cgaacagagtgcggtcagattcacggtttctctcaccttgaacattggat gcaattggattgatccacaatcctggagaatggcttcaagctcactgctc cagtcgcaagcttcagagcctattactaagggtagagctacctatgtcaa gagttttcaaggtacctaagctacatgtgatagtcggcaagccattttga acgcagaccgtgaacggtgatgtaaatccgggatagacgcccaagcgtgc cgtgtcaatgacgctagatacacctcgatttacgtagagtgaatgccagc caatggagtcatgcacataacccgcttagactctgctcggggcgataccc gatcgcagaggcagagccgcttaaacgcgatcgcggtaacctgtaatcag agccagcgctcgatgaattgcatcatggaagccattgatgtggaatgttg agcgtataacaacacgaattgaagacgacattgacttgcttcaagtgagt ggagaattgccgggcagacaagataggtaggctcttggtgcgctgtcaca tcaatccattccttttcctctgttcaatctctatgttgacattctgatag ggatcattggatgccaatgcaaagaacatgagagtgtggtctgcattcaa gtatcctggtcgtaagctgtggccatgggcgctgcggtcaaggtcaatcg cgatgactaatcagtctcggtgactctggggcggtagaggcagtgtcgtg aaccaaagcttgagccgagggcaaaaacaacggcgcatcaaacaatcaac gaaagcatcgtcaacagtgtctcttcccagtcaattacttcgcaaaacct tctcgatagaacccttcagacgatgaacaggccacgcaaccgtcagccgc gccccccaggacagactcagcgcccgggaggcagatcgtcacaccttggt cgacgagctc.

Example 7 Analysis of Select Singly-Substituted CBH2 Variants in Activity and Thermostability Assays

Protoplasts of a six-fold deleted T. reesei strain (Δegl1, Δegl2, Δegl3, Δcbh1, Δcbh2, Δbgl1) were transformed with selected CBH2 SEL variants encoded on the pTTTpyrG vector as described (WO 2009/048488). Spores were harvested, replated on acetamide agar, and incubated at 28° C. for 7 days. Next, CBH2 variants were produced by inoculating 25 ml of YEG culture (5 g/L yeast extract, 20 g/L glucose) with a sporulating Trichoderma strain on agar, followed by a two day incubation period at 28° C., 200 rpm. Five ml of the YEG culture was used to inoculate 45 ml of glycine medium containing a 2% glucose sophorose mixture. The culture was dispensed over a 6-well microtiterplate and incubated stationary for five days at 28° C. in an oxygen-enriched atmosphere. Cells were removed by filtration through a 0.2 μm filter. Supernatants were subsequently concentrated using Vivaspin spin cells with a 10 kD molecular weight cutoff. The variants were tested for their ability to hydrolyze phosphoric acid swollen cellulose (PASC) at 50° C., dilute acid pretreated corn stover (PCS) at 50° C., dilute ammonia pretreated corncob (CC) at 50° C. and 57° C. In addition, the variants were tested for thermostability.

PASC 50° C. Enzyme activity on phosphoric acid swollen cellulose was examined essentially as described in Example 1, Section C, with the following changes. To each well, 10 μl was added containing 4.9 μg protein in supernatant from a CBH2 deleted strain (Δegl1, Δegl2, Δcbh1, Δcbh2). The plate was incubated at 50° C. while shaking at 200 rpm. After 2 hours the plate was put on ice for 5 min and 100 μl of 100 mM glycine pH 10.0 was added. After mixing, the plate was centrifuged at 3000 rpm for 5 min. A volume of 40 μl supernatant was diluted in 160 μl water. Ten μl of the diluted solution was transferred to a new 96-well microtiterplate (Costar Flat Bottom PS) containing 100 μl ABTS glucose assay mixture (2.74 mg/ml 2,2′ azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid, 1 U/ml horseradish peroxidase type VI, 1 U/ml glucose oxidase) and increase in A₄₂₀ was recorded in a microtiterplate spectrophotometer (Spectramax Plus 384, Molecular Devices). A range of glucose concentrations was included as a standard on each plate (0; 0.008; 0.016; 0.031; 0.063; 0.125; 0.25; 0.5; 1 mg/ml). Assays were done in duplicate. A dose response curve was generated for the wild-type CBH2 by fitting the data with a Temkin isotherm equation (y=a+b(ln(1+c*x))) and the activities of the CBH2 variants were divided by a calculated activity of wild-type CBH2 of the same plate to yield a performance index.

PCS 50° C. Enzyme activity on washed dilute acid pretreated cornstover was examined essentially as described in Example 1, Section D, with the following changes. To each well, 10 μl was added containing 49 μg protein in supernatant from a CBH2 deleted strain (Δegl1, Δegl2, Δcbh1, Δcbh2). The plate was incubated at 50° C. while shaking at 200 rpm. After 2 days the plate was put on ice for 5 min and 100 μl of 100 mM glycine pH 10.0 was added. After mixing, the plate was centrifuged at 3000 rpm for 5 min. A volume of 10 μl supernatant was diluted in 190 μl water. Ten μl of the diluted solution was transferred to a new 96-well microtiter plate (Costar Flat Bottom PS) containing 100 μl ABTS glucose assay mixture and assayed and analyzed as described above for the PASC assay.

Corncob 50° C. Enzyme activity on corncob at 50° C. was performed essentially as described in Example 1, Section E, with the following changes. To each well 10 μl solution was added containing 46.55 μg protein of supernatant from a CBH2 deleted strain (Δegl1, Δegl2, Δcbh1, Δcbh2), supplemented with 4.90 μg T. reesei CBH1, 6.84 μg T. reesei Xyn2 Y5 (Xiong et al, Extremophiles 8:393-400, 2004), 2.28 μg Fusarium verticillioides (Fv) 51A, 5.32 μg Fv3A, 0.76 μg Fv43D, and 2.45 μg T. reesei BGL1. The Fusarium verticillioides enzymes have been described in U.S. 61/245,269 (herein incorporated by reference for the teaching of this method). Different volumes of supernatant of a transformed T. reesei strain expressing a CBH2 variant were added as described in Example 1. The plates were incubated for two days at 50° C. with shaking at 200 rpm.

Corncob 57° C. Corn cob was ground to pass through a 0.9 mm screen then pretreated as described in Example 4 of WO 2006/110901 (herein incorporated by reference for the teaching of this method). Pretreated corn cob was used as a 7% cellulose suspension in 50 mM sodium acetate pH 5.0. Seventy microliters of the suspension was added per well to a 96-well microtiterplate (Nunc Flat Bottom PS). To each well 10 μl solution containing 46.55 μg protein of supernatant from a CBH2 deleted strain (Δegl1, Δegl2, Δcbh1, Δcbh2), 4.90 μg CBH1 variant (S8P/T41I/N89D/S92T/S113N/S196T/P227L/D249K/T255P/S278P/E295K/T296P/T332Y/V403D/S411F/T462I), 6.84 μg T. reesei Xyn2 Y5 (Xiong et al, Extremophiles 8:393-400, 2004), 2.28 μg Fv51A, 5.32 μg Fv3A, 0.76 μg Fv43D, 2.45 ug Talaromyces emersonii beta-glucosidase were added. Up to twenty microliters of supernatant from H. jecorina cells expressing either wild-type CBH2 or a CBH2 variant was added. Compensating volumes of acetate buffer were added to make up for differences in total volume. The plate was incubated at 57° C. while shaking at 200 rpm. After 2 days the plate was put on ice for 5 min and 100 μl of 100 mM glycine pH 10.0 was added. After mixing, the plate was centrifuged at 3000 rpm for 5 min. A volume of 10 μl supernatant was diluted in 190 μl water. Ten μl of the diluted solution was transferred to a new 96-well microtiterplate (Costar Flat Bottom PS) containing 100 μl ABTS glucose assay mixture and assayed and analyzed as described above.

CBH2 Thermostability Assay 2. To test the thermostability of the CBH2 variants, 50 μl of supernatant was incubated in a PCR machine across a temperature range of 50-70° C. The remaining activity was determined by incubating 20 μl supernatant with 80 μl 0.625 mg/ml cellotriose in 50 mM sodium acetate buffer pH5.0 with 0.0025% Tween80. After 1 hour, 40 μl of 100 mM glycine pH10 was added. Twenty μl was added to 80 μl ABTS reagent (see above) and color development at OD420 was recorded for 5 min. A pseudo melting temperature (Tm) was calculated by fitting the remaining activity at each temperature with the formula:

y=1/(1+exp(−(a*(1−b/c−ln(c/b))+d*((1/c)−(1/b)))/e))

in which a is the heat capacity (kcal*mol⁻¹*K⁻¹), b is the melting temperature (K), c is the assay temperature (K), d is the enthalpy change (kcal*mol⁻¹*K⁻¹), and e is the gas constant (kcal*mol⁻¹*K⁻¹).

TABLE 7-1 Performance Index and Tm Values for Select CBH2 Variants PASC Variant 50° C. PCS 50° C. CC 50° C. CC 57° C. Tm (° C.)¹ T19K 0.93 0.86 0.99 0.88 55.7 R63L 0.95 0.64 1.05 0.92 56.4 V94A 0.81 0.92 0.89 0.79 56.8 P98Q 1.19 1.07 1.06 0.98 58.8 P98L 0.96 0.52 0.80 0.77 58.3 L111S 0.97 0.96 0.98 1.03 57.8 T138S 1.09 1.02 1.03 1.05 56.5 T138C 1.13 1.09 1.07 1.08 56.3 T138A 0.90 0.93 1.03 0.75 55.6 P143S 0.76 0.69 0.92 0.68 56.5 L144W 0.88 1.07 1.03 0.90 56.5 A150H 1.01 0.95 1.04 1.02 56.9 T154D 0.96 0.97 0.97 0.82 55.9 N161P 0.93 0.88 1.00 0.87 57.0 A177N 0.81 0.81 0.94 0.74 57.3 A177S 0.94 1.41 1.07 0.89 57.1 N182A 0.89 0.80 0.94 0.79 57.4 N182M 1.02 0.97 1.08 1.11 57.5 N182W 0.92 0.33 0.70 0.57 58.8 A188S 1.16 1.30 1.20 1.05 56.4 N197A 1.03 1.16 1.16 1.18 56.1 K288N 0.90 0.80 0.93 0.96 57.4 S291A 1.01 1.03 1.06 1.15 57.1 S291T 0.88 0.73 0.94 0.92 57.0 S291C 1.53 0.95 1.02 1.06 56.3 S291H 1.07 0.87 1.11 1.00 56.6 S291E 0.85 0.61 0.84 0.95 58.0 T312S 1.08 1.07 1.09 1.11 56.7 S313T 1.01 1.08 1.20 1.21 56.4 S316A 1.03 1.03 1.17 1.04 57.0 S316P 1.05 1.09 1.03 1.06 57.1 S316W 1.02 0.94 1.11 1.05 57.0 A322P 1.04 1.05 1.16 1.13 56.7 S343W 0.80 0.60 0.92 1.00 57.1 S343Q 0.94 0.94 1.06 1.16 56.5 S343T 1.09 1.02 1.04 0.82 57.0 S343E 1.10 1.13 1.18 1.12 56.4 F346A 1.04 1.07 1.18 1.14 57.0 F346Y 1.06 1.15 1.06 1.05 56.6 F346H 1.04 0.91 1.04 1.05 57.0 Q362L 1.24 0.99 0.95 1.14 58.1 Q362I 1.02 0.94 0.98 0.93 58.3 Q363S 1.00 1.02 1.08 1.08 57.1 S386D 0.99 0.96 1.00 1.03 55.9 S386A 1.03 1.00 1.06 1.08 55.9 S386F 1.16 0.63 0.94 0.79 52.8 S386I 0.91 0.86 0.98 0.78 56.3 S386C 1.64 1.33 1.10 1.24 56.1 S386V 1.09 0.97 1.04 1.08 56.6 C400S 0.95 0.87 0.93 0.95 64.6 S406P 1.34 1.10 1.06 1.11 57.4 S406A 1.00 1.00 1.02 1.02 57.1 S407T 1.16 1.05 1.08 1.05 57.0 S413W 0.88 0.27 0.66 0.53 59.4 S413Y 0.98 0.91 0.94 1.01 58.0 Q422V 0.97 0.87 1.03 0.93 55.1 Q422T 1.03 1.00 1.08 1.04 55.2 ¹As determined by thermostability assay 2. The Tm of the reference CBH2 of SEQ ID NO: 3 was 57.5° C. under the test conditions.

A category of mutations was devised, that is highly combinable mutations, to encompass mutations that have a PI>0.75 for at least one property, and >0.05 for all properties. As illustrated in the succeeding examples, some variants having two highly combinable mutations, have activity and/or stability that is greater than the reference CBH2 enzyme. An accumulation of highly combinable mutations can be used to make a beneficial variant where each individual mutation is not sufficient to confer superior activity, thermostability and/or expression levels to a variant enzyme.

Example 8 CBH2 Combinatorial Library Variants and Activities Thereof

A synthetic CBH2 combinatorial library was produced by GeneOracle (Mountain View, Calif.). Table 8-1 lists the possible substitutions of members of the CBH2 combinatorial library (numbered according to the CBH2 mature amino acid sequence). This library was created by combining substitutions classified as up mutations and/or highly combinable mutations on the basis of their Tm and performance as listed in Table 7-1.

TABLE 8-1 CBH2 Combinatorial Library Design Targeted Position Wild-Type Residue Substitution 98 P P, Q, L 111 L L.S 182 N N, W 291 S S, E 316 S S, P 362 Q Q, I, L 400 C C, S The library was received from the above-mentioned provider as purified PCR products in which primers GACCGGACGTGTTTTGCCCTTCAT (SEQ ID NO:20) and GTGTGACCGGCTTTGGCGAGTG (SEQ ID NO:21) were used to amplify the cbh2 gene flanked upstream by about 1.1 kb of the cbh1 promoter and downstream by about 1.85 kb of the amdS marker for forced integration in the pyr2 locus in the H. jecorina host strain. A schematic of the homologous recombination of the expression cassette into the screening strain is shown in FIG. 11. The nucleotide sequence of a PCR fragment (partial cbh1 promoter, cbh2 gene, and partial amdS gene) amplified from pTTTpyrG-CBH2 using the primers of SEQ ID NO:20 and SEQ ID NO:21, is provided below as SEQ ID NO:22:

gaccggacgtgttttgcccttcatttggagaaataatgtcattgcgatgt gtaatttgcctgcttgaccgactggggctgttcgaagcccgaatgtagga ttgttatccgaactctgctcgtagaggcatgttgtgaatctgtgtcgggc aggacacgcctcgaaggttcacggcaagggaaaccaccgatagcagtgtc tagtagcaacctgtaaagccgcaatgcagcatcactggaaaatacaaacc aatggctaaaagtacataagttaatgcctaaagaagtcatataccagcgg ctaataattgtacaatcaagtggctaaacgtaccgtaatttgccaacggc ttgtggggttgcagaagcaacggcaaagccccacttccccacgtttgttt cttcactcagtccaatctcagctggtgatcccccaattgggtcgcttgtt tgttccggtgaagtgaaagaagacagaggtaagaatgtctgactcggagc gttttgcatacaaccaagggcagtgatggaagacagtgaaatgttgacat tcaaggagtatttagccagggatgcttgagtgtatcgtgtaaggaggttt gtctgccgatacgacgaatactgtatagtcacttctgatgaagtggtcca tattgaaatgtaaagtcggcactgaacaggcaaaagattgagttgaaact gcctaagatctcgggccctcgggccttcggcctttgggtgtacatgtttg tgctccgggcaaatgcaaagtgtggtaggatcgaacacactgctgccttt accaagcagctgagggtatgtgataggcaaatgttcaggggccactgcat ggtttcgaatagaaagagaagcttagccaagaacaatagccgataaagat agcctcattaaacggaatgagctagtaggcaaagtcagcgaatgtgtata tataaaggacgaggtccgtgcctccctcatgctctccccatctactcatc aactcagatcctccaggagacttgtacaccatcttttgaggcacagaaac ccaatagtcaaccatcacaagtttgtacaaaaaagcaggctccgcggccg cccccttcacccaccatgattgtcggcattctcaccacgctggctacgct ggccacactcgcagctagtgtgcctctagaggagcggcaagcttgctcaa gcgtctggtaattatgtgaaccctctcaagagacccaaatactgagatat gtcaaggggccaatgtggtggccagaattggtcgggtccgacttgctgtg atccggaagcacatgcgtctactccaacgactattactcccagtgtcttc ccggcgctgcaagctcaagctcgtccacgcgcgccgcgtcgacgacttct cgagtatcccccacaacatcccggtcgagctccgcgacgcctccacctgg ttctactactaccagagtacctccagtcggatcgggaaccgctacgtatt caggcaacccttttgttggggtcactccttgggccaatgcatattacgcc tctgaagttagcagcctcgctattcctagcttgactggagccatggccac tgctgcagcagctgtcgcaaaggttccctcttttatgtggctgtaggtcc tcccggaaccaaggcaatctgttactgaaggctcatcattcactgcagag atactcttgacaagacccctctcatggagcaaaccttggccgacatccgc accgccaacaagaatggcggtaactatgccggacagtttgtggtgtatga cttgccggatcgcgattgcgctgcccttgcctcgaatggcgaatactcta ttgccgatggtggcgtcgccaaatataagaactatatcgacaccattcgt caaattgtcgtggaatattccgatatccggaccctcctggttattggTAt gagtttaaacacctgcctccccccccccttcccttcctttcccgccggca tcttgtcgttgtgctaactattgttccctcttccagagcctgactctctt gccaacctggtgaccaacctcggtactccaaagtgtgccaatgctcagtc agcctaccttgagtgcatcaactacgccgtcacacagctgaaccttccaa atgttgcgatgtatttggacgctggccatgcaggatggcttggctggccg gcaaaccaagacccggccgctcagctatttgcaaatgtttacaagaatgc atcgtctccgagagctcttcgcggattggcaaccaatgtcgccaactaca acgggtggaacattaccagccccccatcgtacacgcaaggcaacgctgtc tacaacgagaagctgtacatccacgctattggacctcttcttgccaatca cggctggtccaacgccttcttcatcactgatcaaggtcgatcgggaaagc agcctaccggacagcaacagtggggagactggtgcaatgtgatcggcacc ggatttggtattcgcccatccgcaaacactggggactcgttgctggattc gtttgtctgggtcaagccaggcggcgagtgtgacggcaccagcgacagca gtgcgccacgatttgactcccactgtgcgctcccagatgccttgcaaccg gcgcctcaagctggtgcttggttccaagcctactttgtgcagcttctcac aaacgcaaacccatcgttcctgtaaAagggtgggcgcgccgacccagctt tcttgtacaaagtggtgatcgcgccgcgcgccagctccgtgcgaaagcct gacgcaccggtagattcttggtgagcccgtatcatgacggcggcgggagc tacatggccccgggtgatttattttttttgtatctacttctgaccctttt caaatatacggtcaactcatctttcactggagatgcggcctgcttggtat tgcgatgttgtcagcttggcaaattgtggctttcgaaaacacaaaacgat tccttagtagccatgcattttaagataacggaatagaagaaagaggaaat taaaaaaaaaaaaaaaacaaacatcccgttcataacccgtagaatcgccg ctcttcgtgtatcccagtaccagtttattttgaatagctcgcccgctgga gagcatcctgaatgcaagtaacaaccgtagaggctgacacggcaggtgtt gctagggagcgtcgtgttctacaaggccagacgtcttcgcggttgatata tatgtatgtttgactgcaggctgctcagcgacgacagtcaagttcgccct cgctgcttgtgcaataatcgcagtggggaagccacaccgtgactcccatc tttcagtaaagctctgttggtgtttatcagcaatacacgtaatttaaact cgttagcatggggctgatagcttaattaccgtttaccagtgccatggttc tgcagctttccttggcccgtaaaattcggcgaagccagccaatcaccagc taggcaccagctaaaccctataattagtctcttatcaacaccatccgctc ccccgggatcaatgaggagaatgagggggatgcggggctaaagaagccta cataaccctcatgccaactcccagtttacactcgtcgagccaacatcctg actataagctaacacagaatgcctcaatcctgggaagaactggccgctga taagcgcgcccgcctcgcaaaaaccatccctgatgaatggaaagtccaga cgctgcctgcggaagacagcgttattgatttcccaaagaaatcggggatc ctttcagaggccgaactgaagatcacagaggcctccgctgcagatcttgt gtccaagctggcggccggagagttgacctcggtggaagttacgctagcat tctgtaaacgggcagcaatcgcccagcagttagtagggtcccctctacct ctcagggagatgtaacaacgccaccttatgggactatcaagctgacgctg gcttctgtgcagacaaactgcgcccacgagttcttccctgacgccgctct cgcgcaggcaagggaactcgatgaatactacgcaaagcacaagagacccg ttggtccactccatggcctccccatctctctcaaagaccagcttcgagtc aaggtacaccgttgcccctaagtcgttagatgtccctttttgtcagctaa catatgccaccagggctacgaaacatcaatgggctacatctcatggctaa acaagtacgacgaaggggactcggttctgacaaccatgctccgcaaagcc ggtgccgtcttctacgtcaagacctctgtcccgcagaccctgatggtctg cgagacagtcaacaacatcatcgggcgcaccgtcaacccacgcaacaaga actggtcgtgcggcggcagttctggtggtgagggtgcgatcgttgggatt cgtggtggcgtcatcggtgtaggaacggatatcggtggctcgattcgagt gccggccgcgttcaacttcctgtacggtctaaggccgagtcatgggcggc tgccgtatgcaaagatggcgaacagcatggagggtcaggagacggtgcac agcgttgtcgggccgattacgcactctgttgagggtgagtccttcgcctc ttccttcttttcctgctctataccaggcctccactgtcctcctttcttgc tttttatactatatacgagaccggcagtcactgatgaagtatgttagacc tccgcctcttcaccaaatccgtcctcggtcaggagccatggaaatacgac tccaaggtcatccccatgccctggcgccagtccgagtcggacattattgc ctccaagatcaagaacggcgggctcaatatcggctactacaacttcgacg gcaatgtccttccacaccctcctatcctgcgcggcgtggaaaccaccgtc gccgcactcgccaaagccggtcacacc.

Protoplasts of the AD5 H. jecorina strain (Δegl1, Δegl2, Δcbh1, Δcbh2, Δbgl1) described in Example 6 were transformed with the linear DNA library as described (US 2006/0094080) and grown on selective agar containing acetamide at 28° C. for 7 days (0.6 g/L acetamide, 1.68 g/L CsCl, 20 g/L glucose, 6 g/L KH2PO4, 0.6 g/L MgSO4.7H2O, 0.6 g/L CaCl2.2H2O, 0.5 g/L uridine, trace element salts, 10 g/L low melting point agarose). After 24 hours the agar was overlaid with selective agar supplemented with 1.2 g/L fluoroorotic acid (FOA). A total of 380 colonies were transferred to potato dextrose agar plates containing 1.2 g/L FOA and incubated at 28° C. for 4-5 days. Spores were transferred to fresh potato dextrose agar plates, which were incubated at 28° C. for 3 days. Alternatively, protoplasts of the MAD6 strain described in Example 6 can be employed instead of AD5 for expression of variant library members. Likewise, protoplasts of derivatives of the MAD6 strain in which additional cellulases have been inactivated can be used for this purpose. Such derivatives would exhibit even less background cellulase activity.

For CBH2 variant protein production, spores were transferred using a 96-pin replicator to 200 μl glycine minimal medium supplemented with 2% glucose/sophorose mixture in a PVDF filter plate: 6.0 g/L glycine, 4.7 g/L (NH₄)₂SO₄; 5.0 g/L KH₂PO₄; 1.0 g/L MgSO₄.7H₂O; 33.0 g/L PIPPS; pH 5.5; with sterile addition of a 2% glucose/sophorose mixture as the carbon source, 10 ml/L of 100 g/L of CaCl₂, 2.5 ml/L of T. reesei trace elements (400×): 175 g/L Citric acid anhydrous; 200 g/L FeSO₄.7H₂O; 16 g/L ZnSO₄.7H₂O; 3.2 g/L CuSO₄.5H₂O; 1.4 g/L MnSO₄.H₂O; 0.8 g/L H₃BO₃. Each CBH2 variant was grown in quadruplicate. After sealing the plate with an oxygen permeable membrane, the plates were incubated at 28° C. for 6 days, while shaking at 200 rpm. Supernatant was harvested by transferring the culture medium to a microtiter plate under low pressure.

A total of ten variants that showed improved activity on corn cob at 57° C. were isolated. Genomic DNA of these strains was isolated and their cbh2 gene sequences determined. The CBH2 variants were tested for properties of interest. The substitutions and activities of combinatorial library members on corncob and corn stover is shown in Table 8-2. The specific activities for washed dilute acid pretreated cornstover (PCS 50° C.), for corncob at 50° C. (CC 50° C.), and for corncob at 57° C. (CC 57° C.) were determined as described in Example 7.

TABLE 8-2 CBH2 Combinatorial Variants with Improved Activity on Corncob at 57° C. CC PCS Variant 57° C. CC 50° C. 50° C. CBH2.S291E/Q362I 1.2 0.97 1.13 CBH2.P98Q/S316P/Q362L/L439P 1.52 0.97 0.1 CBH2.P98Q/N182W/S291E/S316P/C400S 1.26 0.97 0.75 CBH2.P98Q/N182W/S291E/S316P/C400S 1.24 0.97 1.02 CBH2.P98L/N182W/S291E/Q362I/C400S 1.26 0.97 1.05 CBH2.P98L/N182W/S291E/Q362I/C400S 1.28 0.97 1.14 CBH2.T74S/P98L/N182W/S291E 1.31 0.97 1.13 CBH2.P98L/N182W/S291E/S316P/Q362I 1.26 0.97 1.22 CBH2.N182W/S291E/Q362L/C400S 1.3 0.97 1.08 CBH2.S291E/Q362L 1.29 0.97 1.18

Example 9 CBH2 Singly and Doubly Substituted Variants and Activities Thereof

Synthetic genes encoding CBH2 variants were constructed by GeneOracle (Mountain View, Calif.) in pTTTpyrG. Table 9-1 lists the single and double variants that were constructed (numbered according to the CBH2 mature amino acid sequence).

TABLE 9-1 Singly and Doubly Substituted CBH2 Variants # Substitution(s) 1. P98Q 2. T138C 3. S316P 4. S343Q 5. Q362I 6. S386C 7. C400S 8. S406P 9. P98Q/T138C 10. P98Q/S316P 11. P98Q/S343Q 12. P98Q/Q362I 13. P98Q/S386C 14. P98Q/C400S 15. P98Q/S406P 16. T138C/S316P 17. T138C/S343Q 18. T138C/Q362I 19. T138C/S386C 20. T138C/C400S 21. T138C/S406P 22. S316P/S343Q 23. S316P/Q362I 24. S316P/S386C 25. S316P/C400S 26. S316P/S406P 27. S343Q/Q362I 28. S343Q/S386C 29. S343Q/C400S 30. S343Q/S406P 31. Q362I/S386C 32. Q362I/C400S 33. Q362I/S406P 34. S386C/C400S 35. S386C/S406P 36. C400S/S406P

The cbh2 variant genes were received from the above-mentioned provider as purified PCR products in which primers GACCGGACGTGTTTTGCCCTTCAT (SEQ ID NO:20) and GTGTGACCGGCTTTGGCGAGTG (SEQ ID NO:21) were used to amplify the cbh2 gene flanked upstream by about 1000 bp of the cbh1 promoter and downstream by about 1000 bp of the amdS marker for forced integration in the pyr2 locus in the H. jecorina host strain. The nucleotide sequence of a PCR fragment (partial cbh1 promoter, cbh2 gene, and partial amdS gene) amplified from pTTTpyrG-CBH2 using the primers of SEQ ID NO:20 and SEQ ID NO:21, is provided above in Example 8 as SEQ ID NO:2q22.

Protoplasts of the MAD6 H. jecorina strain (Δegl1, Δegl2, Δegl3, Δcbh1, Δcbh2, Δbgl1) described in Example 6, were transformed with the linear DNA fragments as described (US 2006/0094080) and grown on selective agar containing acetamide at 28° C. for 7 days as described. After 24 hours the agar was overlain with selective agar supplemented with 1.2 g/L fluoro-orotic acid (FOA). Colonies were transferred to potato dextrose agar plates containing 1.2 g/L FOA and incubated at 28° C. for 4-5 days. Spores were transferred to potato dextrose agar plates, which were incubated at 28° C. for 3 days.

The variants were grown in microtiterplates as described in Example 8. For growth in shake flasks, 25 ml of YEG culture (5 g/L yeast extract, 20 g/L glucose) was inoculated with a sporulating Trichoderma strain on agar and incubated at 28° C. at 200 rpm for 2 days. Next, 5 ml of the YEG culture was used to inoculate 45 ml of glycine medium containing a 2% glucose sophorose mixture in a shake flask. Following inoculation, the cultures were incubated in a resonant acoustic incubator (Applikon) at 28° C. for 3 days. Cells were removed by filtration through a 0.2 μm filter. Supernatants were concentrated using Vivaspin spin cells with a 10 kD molecular weight cutoff.

Samples grown in microtiter plate were analyzed for CBH2 production levels by HPLC as described in Example 1. Both microtiter plate and shake flask grown samples were tested for properties of interest. Table 9-2 lists the performance of the CBH2 variants for multiple properties of interest.

TABLE 9-2 Performance of Singly and Doubly Substituted CBH2 Variants. Variant HPLC PASC PCS CC50 CC57 Tm¹ (° C.) IR² 1 0.53 1.04 1.03 0.98 1.04 57.4 1.11 2 0.76 1.00 1.06 1.03 1.10 50.1 1.06 3 1.01 0.98 1.04 0.99 1.00 55.2 1.40 4 0.98 0.91 0.99 0.94 0.94 56.9 1.05 5 0.97 0.93 1.02 1.03 1.08 56.3 1.20 6 0.48 1.01 1.01 1.02 1.05 54.2 0.89 7 1.07 0.94 0.96 0.95 0.97 64.9 2.29 8 1.02 0.95 1.05 0.99 1.07 58.6 1.22 9 0.53 1.01 1.03 1.00 1.03 59.3 1.19 10 0.63 0.94 0.97 0.96 1.06 59.5 1.50 11 0.56 0.98 0.94 1.05 0.95 57.6 1.12 12 0.50 0.99 1.01 1.15 1.07 58.7 1.39 13 0.19 1.19 0.87 1.02 1.12 50.3 1.21 14 0.96 0.97 1.01 0.97 1.11 59.3 2.39 15 0.66 0.97 0.97 1.00 1.10 57.0 1.40 16 0.84 0.99 1.03 1.03 1.15 59.4 1.60 17 0.78 1.00 1.00 1.04 1.04 52.0 1.45 18 0.72 1.02 1.00 0.98 1.03 57.6 1.28 19 0.40 1.02 1.05 1.01 1.08 56.6 1.08 20 0.94 0.96 1.02 0.92 1.10 59.0 2.30 21 0.83 0.94 0.99 0.96 1.04 54.4 1.37 22 1.04 0.88 0.96 0.92 0.99 47.3 1.26 23 1.00 0.91 0.96 1.00 0.97 60.4 1.48 24 0.55 1.02 1.04 1.07 1.04 58.7 1.30 25 1.15 0.96 0.94 0.87 0.93 64.6 3.19 26 0.89 0.94 0.98 0.98 0.99 59.2 1.67 27 0.95 0.89 0.93 0.95 0.96 55.8 1.33 28 0.47 0.98 0.98 1.00 1.12 56.9 1.27 29 1.12 0.93 1.02 0.93 1.01 62.3 2.57 30 1.00 0.96 0.91 0.95 0.98 60.6 1.23 31 0.47 1.03 1.00 1.00 1.07 60.1 1.37 32 1.06 0.92 0.89 0.96 0.98 65.9 2.90 33 0.99 0.88 0.96 0.97 0.95 60.1 1.60 34 0.88 0.88 0.94 0.98 1.08 58.1 2.41 35 0.55 0.98 1.08 1.03 1.10 59.2 1.24 36 1.09 0.89 0.96 1.00 0.99 65.5 3.19 Wt 1.00 1.00 1.00 1.00 1.00 57.6 1.00 ¹Tm = pseudo melting point temperature (° C.) was determined by thermostability assay 2. ²IR = Performance index for stability as determined by thermostability assay 3.

CBH2 Thermostability Assay 3. To analyze the thermostability of the CBH2 variants, 40 μl of supernatant was incubated in a PCR machine 58° C. in triplicate and samples were removed after 30, 60 and 120 min of incubation. The remaining activity was determined by incubating 10 μl supernatant with 50 μl of 1 mg/ml cellotriose in 50 mM sodium acetate buffer pH 5.0 with 0.0025% Tween20. After one hour, 40 μl of 100 mM glycine pH 10 was added. Fifty μl of 3× concentrated ABTS reagent (see above) was added and color development at OD420 was recorded for 3 min. The residual activity in time was fitted with a formula for exponential decay: y=A₀*exp(−k*t), where A₀ is activity at t=0, t is time, and k is the decay constant. Performance indici (PI) were calculated by the following formula:

PI=−log(k _(variant))/−log(k _(wt)). 

1. A cellulase variant, wherein the variant is a mature form having cellulase activity and comprising a substitution at one or more positions selected from the group consisting of: 5, 18, 19, 28, 30, 32, 35, 38, 79, 80, 89, 100, 102, 103, 104, 105, 111, 117, 119, 121, 125, 126, 133, 137, 138, 139, 140, 141, 143, 150, 158, 162, 177, 180, 181, 182, 185, 186, 188, 190, 191, 192, 193, 196, 201, 207, 225, 226, 228, 229, 230, 233, 234, 236, 240, 243, 245, 251, 252, 258, 267, 268, 274, 292, 293, 303, 304, 306, 307, 313, 319, 322, 328, 331, 338, 340, 346, 361, 362, 363, 364, 365, 371, 384, 394, 396, 400, 406, 407, 414, 417, 422, 427, 431, 433, 436, 440, 441, 443, 444, 445 and 447, wherein the positions are numbered by correspondence with the amino acid sequence of a reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 2. The cellulase variant of claim 1, wherein the variant comprises a further substitution at one or more further positions selected from: (i) a first group consisting of 63, 77, 129, 146, 147, 151, 153, 157, 161, 189, 194, 197, 203, 204, 208, 211, 237, 239, 244, 247, 254, 277, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, 382 and 405; or (ii) a second group consisting of 94, 98, 107, 120, 134, 144, 154, 178, 179, 206, 210, 214, 231, 232, 266, 272, 275, 291, 312, 316, 323, 343, 360, 380, 381, 386, 399, 410, 413, 416, 426, and 429, wherein the further positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 3. A cellulase variant, wherein said variant is a mature form having cellulase activity and comprising a substitution at one or more positions selected from the group consisting of: 63, 77, 129, 146, 147, 151, 153, 157, 161, 189, 194, 197, 203, 204, 208, 211, 237, 239, 244, 247, 254, 277, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, 382 and 405, wherein the positions are numbered by correspondence with the amino acid sequence of a reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not a K/E, R/Q, N/D, Q/E, D/N or E/Q substitution.
 4. The cellulase variant of claim 3, wherein the variant comprises a further substitution at one or more further positions selected from: (i) a first group consisting of 5, 18, 19, 28, 30, 32, 35, 38, 79, 80, 89, 100, 102, 103, 104, 105, 111, 117, 119, 121, 125, 126, 133, 137, 138, 139, 140, 141, 143, 150, 158, 162, 177, 180, 181, 182, 185, 186, 188, 190, 191, 192, 193, 196, 201, 207, 225, 226, 228, 229, 230, 233, 234, 236, 240, 243, 245, 251, 252, 258, 267, 268, 274, 292, 293, 303, 304, 306, 307, 313, 319, 322, 328, 331, 338, 340, 346, 361, 362, 363, 364, 365, 371, 384, 394, 396, 400, 406, 407, 414, 417, 422, 427, 431, 433, 436, 440, 441, 443, 444, 445 and 447; or (ii) a second group consisting of 94, 98, 107, 120, 134, 144, 154, 178, 179, 206, 210, 214, 231, 232, 266, 272, 275, 291, 312, 316, 323, 343, 360, 380, 381, 386, 399, 410, 413, 416, 426, and 429, wherein the further positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 5. A cellulase variant, wherein the variant is a mature form having cellulase activity and comprising a substitution at one or more positions selected from the group consisting of: 94, 98, 107, 120, 134, 144, 154, 178, 179, 206, 210, 214, 231, 232, 266, 272, 275, 291, 312, 316, 323, 343, 360, 380, 381, 386, 399, 410, 413, 416, 426, and 429, wherein the positions are numbered by correspondence with the amino acid sequence of a reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not one of the group consisting of V94E, P98L, E107Q, M120L, M134G, M134L, M134V, L144G, L144R, L144S, T154A, A178V, L179C, V206L, S210L, S210R, T214M, T214Y, G231P, G231I, G231A, G231N, G231S, G231T, T232V, H266S, H266A, W272A, W272D, W272Y, N275L, S316P, V323L, V323N, V323Y, G360R, S380T, A381T, E399N, E399D, R413Y, R413P, and A416G.
 6. The cellulase variant of claim 5, wherein the variant comprises a further substitution at one or more further positions selected from: (i) a first group consisting of 5, 18, 19, 28, 30, 32, 35, 38, 79, 80, 89, 100, 102, 103, 104, 105, 111, 117, 119, 121, 125, 126, 133, 137, 138, 139, 140, 141, 143, 150, 158, 162, 177, 180, 181, 182, 185, 186, 188, 190, 191, 192, 193, 196, 201, 207, 225, 226, 228, 229, 230, 233, 234, 236, 240, 243, 245, 251, 252, 258, 267, 268, 274, 292, 293, 303, 304, 306, 307, 313, 319, 322, 328, 331, 338, 340, 346, 361, 362, 363, 364, 365, 371, 384, 394, 396, 400, 406, 407, 414, 417, 422, 427, 431, 433, 436, 440, 441, 443, 444, 445 and 447; or (ii) a second group consisting of 63, 77, 129, 146, 147, 151, 153, 157, 161, 189, 194, 197, 203, 204, 208, 211, 237, 239, 244, 247, 254, 277, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, 382 and 405, wherein the further positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 7. (canceled)
 8. A cellulase variant, wherein the variant is a mature form having cellulase activity and comprising a substitution at from one to six positions selected from the group consisting of 111, 144, 154, 162, 410 and 413, wherein the cellulase variant comprises: leucine or serine at position 111; leucine, glutamine or tryptophan at position 144; threonine, cysteine or valine at position 154; tyrosine or asparagine at position 162; arginine or serine at position 410; and serine, tryptophan or tyrosine at position 413; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not a S413Y substitution.
 9. A cellulase variant, wherein the variant is a mature form having cellulase activity and comprising a substitution at from one to six positions selected from the group consisting of 98, 194, 313, 316, 384 and 443, wherein the cellulase variant comprises: proline or leucine at position 98; lysine, cysteine or glutamic acid at position 194; serine or threonine at position 313; serine or proline at position 316; glycine, cysteine or glutamine at position 384; and asparagine or isoleucine at position 443; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not one of the group selected from a P98L substitution, a K194E substitution, and a S316P substitution.
 10. A cellulase variant, wherein the variant is a mature form having cellulase activity and comprising a substitution at from one to six positions selected from the group consisting of 153, 161, 203, 233, 422 and 444, wherein the cellulase variant comprises: arginine or glutamine at position 153; asparagine, alanine or tryptophan at position 161; arginine or histidine at position 203; proline or aspartic acid at position 233; glutamine or valine at position 422; and proline or glutamine at position 443; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not a R153Q substitution.
 11. A cellulase variant, wherein the variant is a mature form having cellulase activity and comprising a substitution at from one to seven positions selected from the group consisting of 98, 111, 144, 313, 316, 413 and 422, wherein the cellulase variant comprises: proline or leucine at position 98; leucine or serine at position 111; leucine or tryptophan at position 144; serine or threonine at position 313; serine or proline at position 316; serine or tryptophan at position 413; and glutamine or valine at position 422; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not one of the group selected from a P98L substitution, and a S316P substitution.
 12. A cellulase variant comprising a glutamine at position 98, and a substitution selected from the group consisting of a T138C, a S316P, a S343Q, a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 13. A cellulase variant according to claim 1, comprising a cysteine at position 138, and further comprising a substitution selected from the group consisting of a S316P, a S343Q, a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 14. A cellulase variant comprising a proline at position 316, and a substitution selected from the group consisting of a S343Q, a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 15. A cellulase variant comprising a glutamine at position 343, and a substitution selected from the group consisting of a Q362I, a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 16. A cellulase variant according to claim 1, comprising an isoleucine at position 362, and further comprising a substitution selected from the group consisting of a S386C, a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 17. A cellulase variant comprising a cysteine at position 386, and a substitution selected from the group consisting of a C400S and a S406P, wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3.
 18. The cellulase variant of claim 1, comprising a serine at position 400 and a proline at position
 406. 19. A cellulase variant, wherein the variant is a mature form having cellulase activity and comprising a substitution at from one to seven positions selected from the group consisting of 98, 111, 182, 291, 316, 362 and 400, wherein the cellulase variant comprises: proline, leucine or glutamine at position 98; leucine or serine at position 111; aspargine or tryptophan at position 182; serine or a glutamic acid at position 291; serine or proline at position 316; glutamine, isoleucine or leucine at position 362; and cysteine or serine at position 400; wherein the positions are numbered by correspondence with the amino acid sequence of the reference cellobiohydrolase II (CBH2) set forth as SEQ ID NO:3, and wherein when the cellulase variant consists of a single substitution, the single substitution is not one of the group selected from a P98L substitution, and a S316P substitution.
 20. The cellulase variant of claim 19, wherein the variant further comprises a L439P substitution or a T74S substitution. 21-36. (canceled) 